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PubMed1985

Application of a modified computer algorithm in determining potential antigenic determinants associated with the AIDS virus glycoprotein.

A computer program was developed for use on an Apple IIe that utilized the parameters developed by Hopp and Woods (T. P. Hopp and K. R. Woods, 1983, Mol. Immunol. 20, 483-489) for predicting the hydrophilic regions of a given protein. This program will produce a listing of the hydrophilic sequence averages and graphically illustrates the peak areas. The hydrophilic averages over a hexapeptide length can be used to predict protein structure. In conjunction with the Chou-Fasman predictive scheme for protein secondary structure determination, the possible antigenic determinants for the envelope glycoprotein of three viruses isolated from patients with acquired immunodeficiency syndrome (AIDS) were predicted. These predicted determinants could be used to generate synthetic peptides that represent a potential vaccine preparation or in developing a diagnostic assay that specifically detects the agent.

Acquired Immunodeficiency Syndrome↗

PubMed1978

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. III. The determination of 14C-labeled 6-thiopurines in L5178Y cell extracts using high-pressure liquid cation-exchange chromatography.

A method is presented for the separation of 6-thiopurine bases and ribonucleosides, of sulphate anions and of common purine bases and oxidized purines by means of high-pressure liquid cation-exchange chromatography using a 0.18 X 100 cm column, filled with Beckman M71 resin, and eluted with 0.4M ammonium formate, pH 4.6, at a linear flow velocity of 5.2 cm/min at 50 degrees C. The method has been applied to the separation and quantitative determination of 14C-labeled 6-mercaptopurine metabolites in HClO4 extracts of L5178Y murine lymphoma cells. Distribution patterns of 14C radioactivity within the cells after a 24 h incubation period with (8-14C)-labeled 6-mercaptopurine have been established. The indentification of 6-mercaptopurine metabolites, such as 6-thioxanthosine ribonucleotide, 6-thioinosinic acid, 6-thioguanylic acid, 6-methylthioinosinic acid, and 6-thiouric acid, after the digestion of the extracts with alkaline phosphatase has been confirmed using the behaviour of each compound in enzymatic peak-shifting analyses with purine nucleoside phosphorylase and the corresponding elution volumes of 6-thiopurine bases and ribonucleosides as proofs. According to the specific radioactivity of the (8-14C)-labeled 6-mercaptopurine batch, the amounts of the various 6-mercaptopurine metabolites in about 6% of the total HClO4 extract of 1.6 . 10(8) labeled cells have quantitatively been determined as 1--130 pmol. The intracellular concentration of 6-thiopurines was determined at 1.4 . 10(-5)mol/1.

Alkaline Phosphatase↗

PubMed1990

Repeated determinants within the retinal interphotoreceptor retinoid-binding protein (IRBP): immunological properties of the repeats of an immunodominant determinant.

Interphotoreceptor retinoid-binding protein (IRBP), a glycoprotein which localizes in the retina and pineal gland, induces inflammatory changes in these organs (EAU and EAP, respectively) when injected into various mammals. We have previously identified a determinant (residues 1169-1191) in bovine IRBP which is immunodominant and highly immunogenic and immunopathogenic in Lewis rats. IRBP exhibits a fourfold repeat structure and we report here on the comparison between the active sequence 1179-1191 and its three repeat peptides. Only one of the repeats, 271-283, cross-reacted with 1179-1191 and exhibited immunodominance, albeit of a low level. Peptide 271-283 was also immunogenic and immunopathogenic in Lewis rats, but with a minimal dose approximately 100 times higher than that of 1179-1191. Peptide 880-892, a nondominant determinant, resembled 271-283 in its immunogenicity, but was markedly less immunopathogenic. No immunological activity was detected in the fourth repeat peptide, 579-591. Peptide 1179-1191 was superior to the other repeats also in its antigenicity, i.e., the capacity to stimulate presensitized lymphocytes in culture: the minimal stimulatory concentrations of 1179-1191 was greater than 1000 times lower than those of 271-283 or 880-892. Furthermore, 1179-1191 was stimulatory at concentrations lower than those of 271-283 even when tested with lymphocytes sensitized against 271-283. A correlation was also found between the immunological activities of the repeat peptides and their amphipathicity. This study thus identifies two new immunopathogenic determinants of IRBP and provides additional data to show the association between immunodominance of peptides and their various immunological activities.

Amino Acid Sequence↗

PubMed1983

Determination of human apolipoprotein C-II by electroimmunoassay. Studies on standardization and determination before and after physical training.

1. Human VLDL and HDL were fractionated by sequential ultracentrifugation until free of contaminant plasma proteins. 2. Column chromatofocusing method was used to isolate apolipoprotein C-II from apoVLDL and apo HDL. C-apoprotein peak was rechromatofocused and the second peak was the apo C-II (pI 4.7, homogeneous band on SDS slab gel). 3. New Zealand white rabbits were immunized with apo C-II. Antiserum gave a single precipitate are of identity between whole serum, apoVLDL, apoHDL and apo C-II. 4. Apo C-II concentration was measured by electroimmunoassay method. During standardization 1% Triton X-100 improved the rocket shapes and contours. Total delipidation did not affect the assay system and so the antigenic determinants of apo C-II are all available to antiserum. The lowest concentration of apo C-II possible to determine with this method was 70 ng/sample well. 5. There was no difference between the apo C-II values before (39.8 +/- 7.1 mg/l, n = 19) and after (41.6 +/- 6.4 mg/l, n = 19) moderate physical training among normolipemic subjects. 6. Specific immunoprecipitation technique was also used to determine apo C-II content in standard pool serum.

Adult↗

PubMed1986

Heterogeneity of bovine IgG2--I. The A1 allotypic determinant is the major antigenic determinant recognized on bovine IgG2a by polyclonal rabbit anti-IgG2a.

Rabbit antisera specific for the IgG2a subclass of bovine immunoglobulins also contain antibodies which recognize the A1 allotype of this immunoglobulin subclass. In many sera anti-Al constitutes the major specificity of these rabbit antisera and all of the 10 rabbits immunized with Al(+) IgG2a produced some antibodies which recognized the allotype. The determinant is the same as recognized by a bovine alloantiserum to Al. The Al allotype is shared by the intact and the Fc portion of IgG2a, but rabbits immunized with the Fc fragment also recognize an Al-related determinant which is not exposed on intact Al(+) IgG2a. None of the four rabbits immunized with Al(-) IgG2a produced precipitating antibodies which recognized the gene product of the Al(-), i.e. A2 allele, suggesting that only Al is an immunodominant antigenic determinant for rabbits. Data reported here help to explain the antigenic heterogeneity seen among the IgG2 populations from different cattle when they are tested by immunoprecipitation using rabbit anti-IgG2 reagents.

Animals↗

PubMed2004

Simultaneous determination of 6beta-hydroxycortisol and cortisol in human urine by liquid chromatography with ultraviolet absorbance detection for phenotyping the CYP3A activity determined by the cortisol 6beta-hydroxylation clearance.

This study describes a high-performance liquid chromatographic (HPLC) method for the simultaneous determination of 6beta-hydroxycortisol (6beta-OHF) and cortisol in human urine using either methylprednisolone or beclomethasone as internal standard. Separation was achieved on a reversed-phase phenyl column by a gradient elution of 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH (pH 3.77) and 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH with acetonitrile (4:6, v/v). 6beta-Hydroxycortisol and cortisol were monitored by UV absorption at 239 nm. The lower quantitation limits of the present HPLC method were 21.5 ng/ml for 6beta-OHF and 5.0 ng/ml for cortisol in urine. The within-day reproducibilities in the amounts of 6beta-OHF and cortisol determined were in good agreement with the actual amounts added, the relative error being less than 1.59%. The inter-assay precisions (R.S.D. values) were less than 7.91% for 6beta-OHF and cortisol. The method was compared with the GC/MS method by measuring 6beta-OHF in the same urine samples. A good correlation was found between the amounts determined by the two methods. The regression equations for the HPLC (y) and GC/MS (x) methods were: y=1.0701x+17.389 (r=0.9772) for methylprednisolone as internal standard and y=1.0827x+6.1364 (r=0.9794) for beclomethasone as internal standard.

Anti-Bacterial Agents↗

PubMed2006

Determination of ziprasidone in human plasma by liquid chromatography-electrospray tandem mass spectrometry and its application to plasma level determination in schizophrenia patients.

An accurate, rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay method was developed for the determination of ziprasidone (ZIP) in the plasma of schizophrenia patients. A simple one step liquid-liquid extraction with 20% methylene dichloride in pentane was used to isolate ZIP and the internal standard from the plasma matrix. The compounds were separated on a C-18 column by an isocratic elution and the eluted compounds were analyzed by a triple quadrupole mass spectrometer with a TurboIon spray interface using the positive ion atmospheric pressure electrospray ionization method and detected using multiple reaction monitoring mode. The ZIP standard calibration curve was linear over the range of 0.25-500ng/ml when 0.5ml of plasma was used for the analysis (r(2)>0.998). The intra-assay (within-day) and inter-assay (between-day) variations were less than 12% for the spiked standard curve and quality control samples. The absolute extraction efficiency was 82% for ZIP and 68% for INS-RSP. The analysis time for each sample was less than 3min and useful for high turnaround plasma level determinations. This LC-MS-MS assay method for ZIP is highly specific, sensitive, accurate and rapid and is currently being used for the plasma level determination of ZIP in schizophrenia patients treated with various daily oral doses of ZIP. The data showed large inter-individual variations.

Adult↗

PubMed1999

Determinants of pulmonary venous flow reversal in mitral regurgitation and its usefulness in determining the severity of regurgitation.

Pulmonary venous flow (PVF) reversal is observed in mitral regurgitation (MR) and can be detected by Doppler echocardiography. However, the determinants of PVF alterations in MR have not been analyzed with simultaneous quantitative methods, and the diagnostic accuracy of flow reversal is uncertain. Prospectively, in 128 patients with isolated MR of various degrees (regurgitant fraction 4% to 81%), Doppler echocardiography was used to measure PVF velocity simultaneously to quantify MR by 2 methods and to perform a comprehensive hemodynamic assessment. Systolic PVF velocity was 4 +/- 56 cm/s (systolic flow reversal in 39 patients) and showed the strongest correlations with mitral effective regurgitant orifice (r = -0.56, p <0.0001). In multivariate analysis, larger effective regurgitant orifice (p <0.0001), eccentric jets (p = 0.0023), longer jets (p = 0.0033), and lower mitral regurgitant velocity (p = 0.0015) were independent determinants of decreased systolic PVF velocity. In organic MR, increased filling pressures were associated with systolic PVF reversal. Blunted systolic flow was associated with shorter mitral deceleration time (p <0.0001) and enlarged left atrium (p = 0.0007). For the diagnosis of severe MR (regurgitant orifice > or = 35 mm2, regurgitant fraction > or = 50%), systolic flow reversal sensitivity was 61% and 60%, and specificity was 92% and 85%, respectively. Among 29 patients in whom surgery demonstrated severe mitral lesions, 12 (41%) had no systolic flow reversal preoperatively. In patients with MR, the determinants of systolic PVF are complex and, in addition to the degree of MR, include the hemodynamic consequences of MR, jet characteristics, left ventricular filling, and left atrial volume alterations. Consequently, systolic PVF reversal is a useful sign of severe MR but of relatively low sensitivity, emphasizing the importance of quantifying MR.

Aged↗

PubMed2000

Determination of urinary protein fractions. A comparison with different electrophoretic methods and quantitatively determined protein concentrations.

The second morning urine of 207 patients with various renal diseases was examined. After testing the urine with the Combur-9-RL((R)) dip strip, the concentrations of total protein, creatinine, albumin, alpha-1-microglobulin, immunoglobulin G and transferrin were determined. The urinary protein fractions were measured in parallel with the electrophoresis kits: Hydragel-Proteinuria (sebia GmbH, Fulda); Protur-HiSi (Beckman Coulter GmbH, Krefeld); SPE-II (Beckman Coulter) and the protein concentrations were calculated. The method comparison was based on the agreement of the ascertained types of proteinuria, and on comparison of the electrophoretically obtained albumin protein concentrations with the automated concentration results. It could be shown that urine electrophoresis is a reliable analytic method for the diagnosis of proteinurias. A good agreement and significant correlations were found between single protein determination and urinary electrophoresis. A diagnostic strategy that combines electrophoresis, single protein determination and immunofixation is useful for the diagnosis and differentiation of proteinuria.

Albuminuria↗

PubMed1975

Comparative value of pyruvate determination after glucose loading and transketolase determination for the diagnosis of the Wernicke-Korsakow syndrome.

The determination of transketolase activity and of pyruvic acid after glucose loading in 20 patients with the Wernicke-Korsakow syndrome and in 14 other patients is described. Glucose loading took place after the first injection of thiamine. Normal values for transketolase were found in 10 patients with the Wernicke-Korsakow syndrome, for pyruvic acid after glucose loading in three, for both of these determinations in only one patient. The normal values found for transketolase were probably due in most cases to the administration of thiamine before admission. For practical purposes it is suggested that both determinations be done in all patients with the clinical diagnosis Wernicke-Korsakow's syndrome.

Alcohol Amnestic Disorder↗

PubMed1995

Correlation of total-body bone mineral content determined by dual-energy X-ray absorptiometry with bone mineral density determined by peripheral quantitative computed tomography.

RATIONALE AND OBJECTIVES: We sought to determine the value of peripheral quantitative computed tomography (pQCT) in measuring bone mineral density. METHODS: In 50 healthy, eugonodal premenopausal women, we correlated measurements of total bone mineral content (BMCTB), made with dual-energy X-ray absorptiometry (DXA), and bone mineral density, determined by pQCT. RESULTS: The partial correlations, adjusted for weight and age, between BMCTB and cortical bone density, total bone density, and trabecular bone density were .71 (p < .0001), .63 (p < .0001), and .32 (p < .05), respectively. CONCLUSION: These results and the advantages of pQCT--providing precise bone density determinations for trabecular and compact bone separately, having a high spatial resolution that allows a "compartmental" analysis of bone structure, having a low coefficient of variation, and having a minimal radiation dose (< 5 mrem)--confirm the adequacy of using this method for bone mass studies.

Absorptiometry, Photon↗

PubMed2001

Determination of subyoctomole amounts of nonfluorescent molecules using a thermal lens microscope: subsingle-molecule determination.

The photothermal effect of an ultratrace amount of nonfluorescent molecules in liquid was determined by optimizing the optical arrangement for a thermal lens microscope. The optimized experimental setup could be determined from the evaluation of probing volume and the concentration of the sample solutions even when the expectation of the molecule number in the probing region was less than a single molecule. The minimum expectation, which is explained as being the time average, was 0.4 molecule of Pb(II) octaethylporphyrin (OEP) in benzene. The concentrations in the 9.7 x 10(-11)-7.8 x 10(-10) M region used in this work corresponded to the expected number of 0.4-3.4 molecules, and the calibration curve in this region showed good linearity. Taking into account the enhancement factor of solvent, the molar absorption coefficient of solute, and the optimization of the optical arrangement, the present result, which was the determination limit of 0.34, was consistent with that previously reported. The relation between molecular behavior in the probing volume and the signal was discussed. The average temperature rise in the probing volume by the photothermal effect for the single OEP molecule was estimated as 3.1 muK, and this value was detectable, based on conventional thermal lens measurements for bulk scale sample.

Journal Article↗

PubMed2004

Distributed computing and NMR constraint-based high-resolution structure determination: applied for bioactive Peptide endothelin-1 to determine C-terminal folding.

Distributed computing has been implemented to the solution structure determination of endothelin-1 to evaluate efficiency of the method for NMR constraint-based structure calculations. A key target of the investigation was determination of the C-terminal folding of the peptide, which had been dispersed in previous studies of NMR, despite its pharmacological significances. With use of tens of thousands of random initial structures to explore the conformational space comprehensively, we determined high-resolution structures with good convergences of C-terminal as well as previously defined N-terminal structures. The previous studies had missed the C-terminal convergence because of initial structure dependencies trapped in localized folding of the N-terminal region, which are strongly constricted by two disulfide bonds.

Endothelin-1↗

PubMed2005

Solid state NMR method for the determination of 3D zeolite framework/sorbate structures: 1H/29Si CP MAS NMR study of the high-loaded form of p-xylene in ZSM-5 and determination of the unknown structure of the low-loaded form.

A general protocol is described for structure determinations of organic sorbate-zeolite complexes based on the selective, through-space, distance-dependent transfer of magnetization from protons in selectively deuterated organics to framework silicon nuclei. The method was developed using the known structure of the high-loaded ZSM-5/p-xylene complex containing p-xylene-d(6) or p-xylene-d(4). It was then applied to determine the unknown structure of the low-loaded ZSM-5/p-xylene complex using NMR alone. For the high-loaded complex improved data were obtained below 273 K, where slow motions and exchange processes of the p-xylene are eliminated. The general approach was validated by the exact agreement of the experimental (1)H-(29)Si CPMAS spectra obtained at a specific contact time and the complete 24-line spectra simulated using 1/T(CP) vs M(2) correlations from only the six clearly resolved resonances. For the low-loaded complex the (29)Si resonances were assigned at 267 K, and variable contact time CP experiments were carried out between 243 and 173 K using the same specifically deuterated p-xylenes. All possible locations and orientations of the p-xylene guests were sampled, and those solutions that gave acceptable linear 1/T(CP) vs M(2) correlations were selected. The optimum p-xylene location in this temperature range was determined to be in the channel intersection with the long molecular axis parallel to [0,1,0] (ring center fractional coordinates {-0.009, 0.250, 0.541}) with the ring plane oriented at an angle of 30 +/- 3 degrees about the crystallographic b axis. A subsequent single-crystal X-ray study confirmed this predicted structure.

Deuterium↗

PubMed1975

Determination of fractional renal function in renography with and without individually [131I]albumin-determined blood background.

Fractional renal function was determined from 122 renographies using three different blood background subtractions: with individually [131I]albumin-determined correction factors, with direct subtraction of the thorax background curve. In the latter case a systematic error was introduced, since the right kidney's fractional function was overestimated and that of the left kidney accordingly underestimated. With mean correction factors the deviation from 'true' fractional function (as determined individually with [131I]albumin) was negligible. We recommend this principle since it is simple and sufficiently accurate.

Adult↗

PubMed1978

Determination of net protein utilization using whole carcass, hind leg or liver of the rat and its relationship with protein efficiency ratio determination.

Experiments were conducted to test the simplified NPU method designed by Lachance and Miller with diets of different protein quality and to determine the NPU using the liver nitrogen concentration instead of that in the hind limb. The NPU determination was done after 10- and 21-day periods instead of a 28-day period. The second part of the study was the analysis of the relationship between NPU and PER done with the same rats. The NPU calculated from whole carcass nitrogen concentration showed a high correlation with the NPU calculated from the nitrogen concentration in the leg (r = 0.990) and also with the NPU calculated from the liver nitrogen concentration. The Tukey's "t" test showed that in the 10-day experiment discrimination between the effect of the different diets upon NPU was not as good as was the case at 21 days. However, the NPU values at 10 days were in the same sequence as those at 21 days. For practical purposes it is more reliable and easier to perform the NPU determination using the liver instead of the whole carcass or leg. The statistical analysis of PER and NPU showed the same discriminating value when the test diets were isoenergetic.

Animals↗

PubMed1992

Ab initio phase determination for spherical viruses: parameter determination for spherical-shell models.

The structure determination of canine parvovirus depended on the extension of phases calculated initially from a spherical-shell model [Tsao, Chapman, Wu, Agbandje, Keller & Rossmann (1992). Acta Cryst. B48, 75-88]. Such ab initio phasing holds the promise of obviating initial experimental phasing by isomorphous or molecular replacement, thereby expediting the structure determinations of spherical virus capsids. In this paper, it is shown how parameters such as radii, DNA density and particle positions may be determined and refined from diffraction data with sufficient precision to start phase extension from 20 A resolution for a virus of approximately 122 A radius.

Capsid↗

PubMed1998

STEP--a trial-and-error procedure for crystal structure determination. II. The determination of two small protein structures.

This paper describes the difficulties in the process of using the trial-and-error SYSTEM90 program to determine ab initio the structures of two small proteins App [Woolfson & Yao (1990). Acta Cryst. A46, 409-413] and rubredoxin [Sheldrick et al. (1993). Acta Cryst. D49, 18-23] with high-resolution data. Some strategies for overcoming the difficulties are discussed and the upgraded SYSTEM95 program was used successfully to determine the two structures. The most characteristic feature of this structure-determination process is that the two proteins are treated as unknown structures with only their chemical compositions and high-resolution data sets known. A new figure of merit R(sc), replacing the old figure of merit, XDFOM, is quite effective in picking out a good set of phases in the multi-solution stage when the phases are overconsistent. Controlling the Fourier recycling technique and the residuals can separate the mixture of structures and the enantiomorph and finally give one absolute structure. The results are compared with known structures to verify their reliability.

Animals↗