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PubMed1983

T cell allotypic determinants encoded by genes linked to the immunoglobulin heavy chain locus. I. Establishment of monoclonal antibodies against allotypic determinants.

Monoclonal alloantibodies for T cell allotypic determinants were obtained by hybridizing SP-2 tumor cells with BALB/c (H-2d, Igh-1a) spleen cells, which had been repeatedly immunized with Con A-stimulated CB-20 (H-2d, Igh-1b) spleen cells. It was found that these monoclonal anti-CB-20 antibodies detect the new allotypic determinants (distinct from the B cell Igh-C region determinant) expressed only on the augmenting or suppressor T cells. Genetic analysis of these antigenic determinants revealed these antibodies react with the gene products located on the telomeric side chromosome of the Igh variable region gene (Igh-V) cluster. These antibodies when given in vivo caused a modification of antibody production. The antibody activity was absorbed by Con A-stimulated B10.BR (H-2b, Igh-1b), C57BL/6 (H-2b, Igh-1b), CWB (H-2b, Igh-1b), CB-20 (H-2d, Igh-1b), and BAB-14 (H-2d, Igh-1b) spleen cells, but not by Con A-stimulated C3H.SW (H-2b, Igh-1j), BALB/c (H-2d, Igh-1a), A/Sn (H-2a, Igh-1e), and C.AL-20 (H-2d, Igh-1d) spleen cells. In addition, in vivo these monoclonal antibodies modified anti-SRBC antibody production only in Igh-1b allotype-bearing mice. One monoclonal antibody reacted with 4-hydroxy-3-nitrophenyl acetyl- (NP) hapten-specific augmenting T cells, and the other two batches of monoclonal antibodies reacted with NP-specific suppressor T cells of NP-mediated cutaneous responses. A mapping study with these recombinants limits the gene coding for the T cell-specific determinants to a gene within the variable region to the telomeric side of NP-VH and to the centromeric side of prealbumin. This segment is inclusive of all immunoglobulin genes, the region Owen named IgT-C, and a histocompatibility gene (H-Ig).

Animals↗

PubMed1994

[Determination of ascorbic acid in blood serum using high-performance liquid chromatography and its correlation with spectrophotometric (colorimetric) determination].

Insufficient supply of the populations of the Czech and Slovak Republics with vitamin C is connected with the occurrence of some diseases due to modern civilization. It is therefore necessary to obtain reliable data about the supply of the population with this most important antioxidant of blood plasma. In order to establish a simple, rapid and at the same time specific determination of vitamin C in serum, the present authors have introduced, on the basis of foreign data, the HPLC method of determination of ascorbic acid on reverse phases with UV-detection (245 nm), using 3.7 mM KH2PO4 (pH = 4) as the mobile phase. Very good separation of ascorbic acid from serum (Fig. 1) was obtained. The detection limit is 3.25 pmol of ascorbic acid. Parallelly with the chromatographic determinations, ascorbic acid was determined in 235 volunteers also by the spectrophotometric (colorimetric) method employing 2,4-dinitrophenylhydrazine. Both methods correlate very well (Fig. 2): correlation coefficient, r = 0.93, the level of probability p < 0.000001. The yield (Table 1) is 98.6% and 97.6% for HPLC, or 105.4% and 111.4% for the colorimetric method. The reproducibility of both methods gives coefficients of variation of 3.6% and 2.9%. The described HPLC method is sufficiently precise, specific and suitable for the determination of the levels of ascorbic acid in serum.

Adult↗

PubMed1996

Determinants of weddellite formation: chondroitin sulfates and citrate determine weddellite formation in vitro.

In synthetic urine (SU), addition of oxalate tends to form monohydrates of calcium oxalate. However, addition of oxalate to natural urine preferably forms calcium oxalate dihydrate (COD). Urine apparently contains a determinant for COD formation. To identify the determinant, the effects of pH, temperature, oxalate, calcium, urate, citrate, magnesium, sulfate and chondroitin sulfates (CS) on calcium oxalate crystal formation were studied. Lower temperatures, higher oxalate concentrations and higher pH favored COD formation in a SU. Mixed CS in the presence of citrate were the most decisive determinant of COD formation. Substitution of CS for agar and gelatin produced similar results, indicating that the colloidal effect of the macromolecules determines COD formation. Identification of the determinants led to a simple, reproducible method of COD formation in SU without natural urine. Addition of strontium to SU resulted in dodecahedral bipyramids. Interpenetration twinning of bipyramids occur within seconds of the crystal formation.

Calcium Oxalate↗

PubMed1994

Role of reductase and aromatase in sex determination in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination.

In many turtles the temperature during the middle of incubation determines the gonadal sex of the hatchling. In the red-eared slider turtle (Trachemys scripta), an incubation temperature of 26 degrees C results in all male offspring, whereas an incubation temperature of 31 degrees C results in all female offspring; at temperatures intermediate to these (e.g. 29, 29.2, 29.4 degrees C) a mixed sex ratio is obtained. Administration of exogenous oestrogens will overcome the effects of an all-male producing incubation temperature to cause female sex determination, whereas administration of exogenous dihydrotestosterone (DHT) or testosterone to eggs incubating at an all-female temperature will have no discernible effect. Administration of DHT will cause male sex determination only if administered at intermediate incubation temperatures whereas administration of testosterone to eggs incubating at all male-producing and male-biased intermediate temperatures results in a significant number of female offspring, an effect presumably due to aromatization of testosterone to oestradiol (OE2). Since testosterone serves as the precursor to both DHT and OE2, being metabolized by reductase and aromatase respectively, three experiments were conducted to determine whether various putative reductase and aromatase inhibitors would overcome the effect of incubation temperature. First, while administration of testosterone to eggs incubating at all male-producing and male-biased intermediate temperatures produced females in a dose- and temperature-dependent manner, significant numbers of intersex individuals resulted from high dosage testosterone treatment to eggs incubating at a female-biased intermediate temperature. The reductase inhibitors 4MA and MK906 were capable of producing female offspring if administered at intermediate temperatures, but not in a dose-dependent fashion. Administration of the aromatase inhibitors CGS16949A and CGS20267 resulted in male offspring at both female-biased intermediate and at all female-producing temperatures in a dose-dependent fashion. Second, similar findings were obtained with combined doses of testosterone and reductase or aromatase inhibitors. Combined treatment of eggs at male-biased intermediate incubation temperatures with testosterone and reductase inhibitor resulted in female hatchlings, whereas combined treatment of testosterone and aromatase inhibitor at both female-biased intermediate and at all female-producing temperatures resulted in male hatchlings.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Radioligand exchange binding cannot directly determine the dissociation constant (Kd) of the rat ventral prostate nuclear androgen receptor: valid Kd determinations require additional uptake binding data.

Radioligand exchange was examined for its ability to derive the dissociation constant (Kd) of the rat ventral prostate nuclear androgen receptor. In one 24-h, 12 degrees C incubation, Scatchard plot analysis of [3H]dihydrotestosterone ([3H]DHT) exchange binding produced a Kd of 6.9 x 10(-9) M. Specific binding of [3H]DHT ranged from 114 to 758 pM, and the extrapolated value for the total number of binding sites (n) was 1320 pM. When aliquots from the same receptor pool were incubated with unlabeled DHT, and bound androgen was measured by radioimmunoassay, each titration point held a concentration of specifically bound unlabeled DHT little different from the preincubation value of bound endogenous ligand (1338 pM), suggesting that few, if any, unoccupied sites were created during the incubation. In a second radioligand exchange assay, unoccupied receptor sites were measured at the end of incubation. Virtually no unoccupied sites were found, though the range of predicted values was 124 to 383 pM (n = 425 pM). The data, in toto, suggest that although radioactive ligand exchanges with bound unlabeled ligand, the dynamics of the process do not include the creation of unoccupied sites. Since the Kd is determined by measuring the concentrations of unoccupied sites, free ligand, and receptor sites bound to ligand, the absence of unoccupied sites suggests that radioligand exchange cannot be used to directly determine the Kd of the prostate nuclear androgen receptor. The numerical value obtained from radioligand exchange, therefore, instead of being a Kd, is very likely the result of a graphic plot of the increase in specific activity of bound radioligand as [3H]DHT is titrated to higher levels. In the last phase of the study a technique was developed which allows for the correct determination of the Kd of the rat ventral prostate nuclear androgen receptor. For the determination, data from an experiment measuring uptake binding into unoccupied sites were combined with data obtained from radioligand exchange binding. From this, the Kd of the receptor was calculated to be 1 x 10(-12) M.

Animals↗

PubMed1986

[Determination of rotational defects of the femur by computer tomographic determination of the antetorsion angle of the femoral neck].

The first of all unexplained but persisting static complaints after femoral fractures are due to rotational deformities. The clinical examination can only sustain the suspicion of a rotational deformity but can not determine its exact extent. The usual X-ray determination of the anteversion angle in the technique of Dunn and Rippstein as a measure for torsional deformities is often very difficult, because of the exact positioning conditions of patients with posttraumatic axial deformities, soft tissue contractures and painful motility restrictions. With the total body computertomography we have a safe and quick technique, independent of the patient's positioning, to determine the anteversion angle without problems. In comparing the two sides, the rotational deformity can be recognized and its extent can exactly be measured. Because of the simple technique and the exact determination of rotatory deformity a computertomographic measuring of the anteversion of the femoral head always should be made before planning a corrective osteotomy to fix the extent of correction, whenever a total body scan is available.

Adult↗

PubMed1994

Relation of coronary calcium determined by electron beam computed tomography and lumen narrowing determined by autopsy.

Coronary calcium determined by electron beam computed tomography (CT) has not been systematically evaluated regarding prediction of histopathologic atherosclerotic disease. Furthermore, gender specificity has not been examined. The 3 major epicardial arteries were dissected from 13 consecutive hearts (5 women and 8 men) after autopsy. Each artery was straightened and scanned using CT in contiguous 3 mm thick cross sections. After imaging, histologic sections were prepared at corresponding intervals and luminal area obstruction determined by planimetry. Electron beam CT scans were analyzed to determine coronary calcium area (i.e., tomographic area with CT density > 130 Hounsfield units). A total of 522 histologic specimens were examined and paired with corresponding CT scans (182 in women, 340 in men). Receiver-operating characteristic (ROC) analysis was used to define site specificity of calcium area for luminal area narrowing by atherosclerosis. ROC curve areas for segmental CT calcium and prediction of atherosclerosis representing mild, moderate, or severe disease were, respectively, 0.712, 0.843, and 0.857 for women and 0.732, 0.793, and 0.841 for men. Curves relating false-positive rate (1-specificity) to predefined degrees of atherosclerotic narrowing versus calcium area were curvilinear. In both women and men, calcium areas on the order of 1 mm2/coronary segment were necessary to predict at least mild atherosclerosis with a false-positive rate of 0% (i.e., 100% specificity), whereas a calcium area > 3 mm2 was necessary to predict the same result for severe disease. In conclusion, coronary artery calcium area as determined by electron beam CT has the potential to predict segmental histopathologic coronary disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1978

A new method for allantoin determination and its application in allantoin determination in Agrostemma githago L. Seed.

We have established several optimal conditions for qualitative and quantitative allantoin determination by applying Ehrlich's reagent. The limit of detection for allantoin determination amounts to 5 x 10(-6) mM. Allantoin is determined quantitatively by measuring the absorbance at 440 nm (from 300 to 1000 micrograms/ml). The color of the complex becomes stable by standing for 10 min at room temperature. We have used these conditions for allantoin determination in Agrostemma githago seed.

Allantoin↗

PubMed1992

Determination of IgE antibodies to the benzyl penicilloyl determinant. A comparison between poly-L-lysine and human serum albumin as carriers.

The influence of two different carriers, poly-L-lysine (PLL) and human serum albumin (HSA) in the binding of specific IgE antibodies to the benzyl penicilloyl hapten (BPO) was determined in a solid-phase assay. Serum samples from patients with a history of immediate reaction to penicillin and which had shown the presence of IgE antibodies to BPO were used. Benzyl penicilloyl derivatized cellulose discs were prepared using PLL of different molecular weight and HSA as carriers. These were treated with different molar ratios of benzyl penicillin. These reagents were compared for uptake of BPO-specific IgE using a pool of sera in a radioallergosorbent test (RAST) type assay. Two PLL systems and two HSA systems were finally compared using 26 individual sera. RAST values were compared by Kruskal-Wallis and Wilcoxon tests. The relationships between the four different assays were evaluated by determining Pearson correlation coefficients and the concordance by determining intraclass correlation coefficients (ICC). Analysis of means by the Wilcoxon test revealed significant differences (P less than 0.01) only when the different carrier assays were compared. The correlation coefficients between all the assays were significant (P less than 0.0001), but the ICC was low when the different carrier assays were compared. These results indicate that the nature of the carriers studied (PLL and HSA) influences the capacity for binding IgE antibodies in the RAST procedure. The differences observed indicate that one conjugate cannot be substituted for the other in the determination of IgE antibodies to BPO and that BPO-PLL is preferable.

Antibodies↗

PubMed1977

Hypocomplementaemic and normocomplementaemic multiple sclerosis. Genetic determinism and association with specific HLA determinants (B18 and B7).

Seventy-five patients with multiple sclerosis (MS) were treated for complement components C3, after factor B, C4, and tested for HLA-A and B-determinants. Levels of IgG, IgA, IgD, IgE and titres of measles antibodies were also determined. Correlations between these immunological values and HLA determinants could be obtained in siblings, parents and/or children of the patients in 13 families. B18 frequency is strongly associated with the hypocomplementaemic group (x2 = 8.9). An association of B18 with the population of cases with low B levels is also found (x2 = 8.02). Familial data showed that low C3 and/or low B levels are associated with the HLA haplotyes, especially with those containing B18. A "complement abnormality susceptibility gene", linked to the HLA genes, is postulated. Infections are significantly more frequent in families of hypocomplementaemic MS, the existence of a genetic immunodeficiency affecting the synthesis of the complement components, linked to the HLA determinants. In 1 case studied in this article, a heterozygous C2 deficiency linked to HLA-A10, B18 was found and might confirm this hypothesis.

Antibodies, Viral↗

PubMed1995

Determination of the concentration of influenza virus antihaemagglutinin antibody molecules of the IgG class and of the equilibrium constant by use of enzyme immunoassay titres determined for graded epitope concentrations.

A new technique for determining the concentration of influenza virus antihaemagglutinin antibody molecules of the IgG class (A) and of the equilibrium constant K of paratope-epitope interaction is described. The method is based on determining enzyme immunoassay (EIA) titres of antibody with graded epitope concentrations: EIA titres were defined in terms of the antibody dilution yielding a fixed amount of antibody adsorbed per ml (= 6.21 x 10(10)). Adsorption of antibody depends on the concentration of paratopes and epitopes allowed to react and on the equilibrium constant. For the use of a constant concentration of epitopes, the paratope concentration needed to yield the desired degree of antibody adsorption decreases with increasing avidity. Therefore, the EIA titres increase both with increasing avidity and increasing antibody concentration. When graded epitope concentrations are used for determining the EIA titres of a given serum, the titres are influenced in a similar manner by the antibody concentration of the serum and the increase of titres with increasing epitope concentration reflects avidity. The equilibrium constant found is subsequently used to determine the concentration of free antibody at the dilution meeting the definition of EIA titre and the product of EIA titre and the sum of free and bound antibody at this dilution gives the number of antibody molecules present in the test serum. A panel of 118 antisera was tested comparatively for A and K using the new method and by means of the guanidine titre ratio test and equilibrium filtration. The values obtained agreed well with each other. This novel technique offers the advantage that it can be easily adapted for use with other viruses.

Animals↗

PubMed2006

Determination of trace levels of total fluorine in water using combustion ion chromatography for fluorine: a mass balance approach to determine individual perfluorinated chemicals in water.

Perfluorinated compounds (PFCs) such as perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) have received worldwide attention because of their environmental persistence and widespread distribution. Because of the lack of robust analytical methods and standards to detect all of the PFCs, and their precursors and metabolic intermediates, a mass balance approach involving the determination of total fluorine (TF), followed by fractionation of samples to separately determine inorganic and organic fluorine, is needed. In this study, we have developed a method to determine low microg/L levels of total fluorine (TF) in seawater samples. Further, seawater samples were fractionated into organic and inorganic fractions by extraction with organic solvents, which were then analyzed for TF, extractable organic fluorine (EOF) and inorganic fluorine (IF; i.e., fluoride). Concentrations of known perfluorinated compounds (PFCs) including PFOS and PFOA were also determined in water samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to enable calculation of the fraction of fluorine that is contributed by PFCs to TF. A major proportion of fluorine in seawater was in the form of fluoride (>90% in locations not affected by direct discharges). Nevertheless, within the organofluorine fraction, a major percentage (60-90%) of fluorine still remains unknown in water samples, suggesting the occurrence of other fluorinated acids in addition to known perfluorinated acids. Further studies are needed to identify and quantify the unidentified organofluorines in seawater. Mass balance analysis of total organic fluorine (TOF) and EOF is important, if we are to understand transport and fate of fluorinated compounds in the environment, and if we are to identify the sources of unidentified fluorinated compounds.

Chromatography, Liquid↗

PubMed1978

The renal perfusion/excretion determination renogram: a new method of individual renal function determination in obstructive renal disease.

The divided renal function of 10 patients with unilateral renal obstruction was measured with the renal perfusion/excretion determination renogram, a computerized radioisotope function study, and with endogenous creatinine clearances. Functional impairment of the obstructed kidney when estimated by the effective renal plasma flow determination of the renal perfusion/excretion determination renogram correlated closely with that measured by creatinine clearance. The clinical applications of the renal perfusion/excretion determination renogram in unilateral renal obstruction include the preoperative evaluation of individual renal function and serial estimates of individual renal function in chronic obstructive processes.

Adult↗

PubMed1998

Determination of C1-C5 alkyl nitrates in rain, snow, white frost, lake, and tap water by a combined codistillation head-space gas chromatography technique. Determination of Henry's law constants by head-space GC.

Alkyl nitrates with a chain length up to five carbon atoms have been determined in snow, white frost, and surface water. The samples were taken in the vicinity of Ulm, Germany, a region in central Europe. The determination of C1-C5-alkyl nitrates in water samples was achieved with a new water codistillation enrichment technique directly coupled with on-column head-space gas chromatography. The concentrations of the short chain alkyl nitrates in the different forms of wet deposition range from 89 ng L-1 for 1-propyl nitrate down to 35 ng L-1 for 1-pentyl nitrate. C1-C5-alkyl nitrates in wet depositions were also directly determined by static head-space gas chromatography. Gas-water partition coefficients KGW (Henry's law constant H) were determined by head-space gas chromatography and secondly by calculating the Henry's law constant by the ratio of vapor pressure to water solubility. The gas-water partition constants (dimensionless) or Henry's law constants range from KGW = 0.038 (H = 93 Pa m3 mol-1) for 1-propyl nitrate up to KGW = 0.122 (H = 302 Pa m3 mol-1) for 2-pentyl nitrate.

Chromatography, Gas↗

PubMed2000

Chain length determination of prenyltransferases: both heteromeric subunits of medium-chain (E)-prenyl diphosphate synthase are involved in the product chain length determination.

Among prenyltransferases, medium-chain (E)-prenyl diphosphate synthases are unusual because of their heterodimeric structures. The larger subunit has highly conserved regions typical of (E)-prenyltransferases. The smaller one has recently been shown to be involved in the binding of allylic substrate as well as determining the chain length of the reaction product [Zhang, Y.-W., et al. (1999) Biochemistry 38, 14638-14643]. To better understand the product chain length determination mechanism of these enzymes, several amino acid residues in the larger subunits of Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase and Bacillus subtilis heptaprenyl diphosphate synthase were selected for substitutions by site-directed mutagenesis and examined by combination with the corresponding wild-type or mutated smaller subunits. Replacement of the Ala at the fifth position upstream to the first Asp-rich motif with bulky amino acids in both larger subunits resulted in shortening the chain lengths of the major products, and a double combination of mutant subunits of the heptaprenyl diphosphate synthase, I-D97A/II-A79F, yielded exclusively geranylgeranyl diphosphate. However, the combination of a mutant subunit and the wild-type, I-Y103S/II-WT or I-WT/II-I76G, produced a C(40) prenyl diphosphate, and the double combination of the mutants, I-Y103S/II-I76G, gave a reaction product with longer prenyl chain up to C(50). These results suggest that medium-chain (E)-prenyl diphosphate synthases take a novel mode for the product chain length determination, in which both subunits cooperatively participate in maintaining and determining the product specificity of each enzyme.

Alkyl and Aryl Transferases↗

PubMed1997

Protein phi and psi dihedral restraints determined from multidimensional hypersurface correlations of backbone chemical shifts and their use in the determination of protein tertiary structures.

The chemical shifts of the backbone atoms of proteins can be used to obtain restraints that can be incorporated into structure determination methods. Each chemical shift can be used to define a restraint and these restraints can be simultaneously used to define the local, secondary structure features. The global fold can be determined by a combined use of the chemical shift based restraints along with the long-range information present in the NOEs of partially deuterated proteins or the amide-amide NOEs but not from such limited NOE data sets alone. This approach has been demonstrated to be capable of determining the overall folding pattern of four proteins. This suggests that solution-state NMR methods can be extended to the structure determination of larger proteins by using the information present in the chemical shifts of the backbone atoms along with the data that can be obtained on a small number of labeled forms.

Amino Acids↗

PubMed2002

Sensitive determination of low molecular mass phenols by liquid chromatography with chemiluminescence detection for the determination of phenol and 4-methylphenol in urine.

A rapid, reliable method for the routine determination of phenol and 4-methylphenol in urine samples by liquid chromatography with peroxyoxalate chemiluminescence detection was developed. Phenols were first cleaned up by passing the sample through a LiChrolut EN sorbent column and then derivatized straightforwardly with dansyl chloride (15 min at room temperature) thanks to the micellar catalytic effect provided by Triton X-100 micelles. The derivatives were successfully separated in 15 min on a C18 analytical column and determined using an integrated derivatization chemiluminescence detection unit based on the bis(2,4,6-trichlorophenyl) oxalate-hydrogen peroxide system. Linear ranges from 3 to 500 microg L(-1), limits of detection at a signal-to-noise ratio of 3 from 0.3 to 0.5 microg L(-1) and relative standard deviations from 2.8 to 4.7% were obtained. The proposed method was applied to the assay of different human urine samples (healthy, smoker and petrol station worker volunteers) and free and total phenol and 4-methylphenol were determined. The proposed method surpasses other chromatographic alternatives for the determination of these phenols in terms of limit of detection and sample requirements for the analysis.

Chromatography, Liquid↗

PubMed1984

[Determination of left atrial size. Comparison between echocardiographic and radiological determination].

The size of the left atrium was determined echocardiographically and radiologically in 66 patients with mitral valve disease, grade I-IV (New York Heart Association classification). Angiocardiography served as the reference. The echocardiographic determination was both by M-mode and two-dimensional, the radiological one was from the postero-anterior films (after Higgins), as well as from the lateral pictures. There was good agreement between the two echocardiographic methods and a good correlation of the two with cine-angiographic measurements (r = 0.82-0.99; P less than 0.001). But there was a poorer, although still statistically significant correlation of the radiologically determined atrial size with the echocardiographic and angiocardiographic measurements (r = 0.47-0.56; P less than 0.001). Thus M-mode and two-dimensional echocardiography is the more sensitive method for determining the size of the left atrium.

Adult↗