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CXC chemokines bind to unique sets of selectivity determinants that can function independently and are broadly distributed on multiple domains of human interleukin-8 receptor B. Determinants of high affinity binding and receptor activation are distinct.

Human interleukin-8 receptors A (IL-8RA) and B (IL-8RB) are seven-transmembrane domain (TMD) neutrophil chemokine receptors with similar sequences (77% amino acid identity) and similar G protein selectivity, but markedly different selectivity for CXC chemokines. IL-8RB is selective for IL-8, growth-related oncogene alpha (GRO alpha) and neutrophil-activating peptide-2 (NAP-2), whereas IL-8RA is selective only for IL-8. To identify selectivity determinants, we made eight chimeric receptors exchanging: 1) the three main regions of sequence divergence between IL-8RA and IL-8RB (the N-terminal segment before TMD1, the region from TMD4 to the end of the second extracellular (e2) loop, and the C-terminal tail), and 2) the N-terminal segment of CC chemokine receptor 1, which does not bind CXC chemokines. Chimeras were tested by direct 125I-IL-8, 125I-GRO alpha, and 125I-NAP-2 binding, heterologous competition binding, and calcium flux assays using human embryonic kidney 293 cells stably transfected with receptor DNAs. The following results were obtained: 1) chimeric receptors had binding sites for IL-8, GRO alpha and NAP-2 distinct from those on IL-8RA and IL-8RB; 2) IL-8, GRO alpha and NAP-2 bound to overlapping but distinct sites that mapped differentially to multiple domains on IL-8RB; 3) high affinity radioligand binding and high agonist potency were separable functions for IL-8, GRO alpha and NAP-2, suggesting that the determinants of high affinity binding may not be critical for receptor activation; and 4) determinants of GRO alpha and NAP-2 selectivity were found in both the N-terminal segment before TMD1 and the region from TMD4 to the end of the e2 loop of IL-8RB, and functioned independently of each other. Stated reciprocally, the N-terminal segment of IL-8RA was not a dominant selectivity determinant. These data suggest that both narrow and broad spectrum chemokine antagonists can be developed to block functions mediated by IL-8RB.

Amino Acid Sequence↗

Determination of ITPase activity in erythrocyte lysates obtained for determination of TPMT activity.

The indication for the determination of both thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphohydrolase is identical (i.e., adverse drug reactions toward mercaptopurines). Therefore, we tested whether or not our standard procedure to prepare erythrocyte lysates for measurement of TPMT activity, which includes treatment with Chelex 100 (a chelating resin), was suitable for the measurement of ITPase activity. It also was tested to see if ITPase activity differs in EDTA and Heparin anti-coagulated blood samples. We found that there was no difference between the ITPase activity in erythrocyte lysates prepared from EDTA or Heparin anti-coagulated blood. Treatment with a chelating resin or omission of magnesium from the assay procedure resulted in decreased and nearly absent ITPase activity, respectively. We conclude that untreated erythrocyte lysates obtained for determination of TPMT activity are suitable for determination of ITPase activity. However, after treatment with Chelex 100 the erythrocyte lysates become unsuitable for determination of ITPase activity.

Chelating Agents↗

Viral determinants of HIV-1 sufficient to extend tropism to macrophages are distinct from the determinants that control the cytopathic phenotype in HL-60 cells.

DESIGN: Infection of the human promyelocytic cell line HL-60 with NL4-3, a molecularly cloned HIV-1 strain that productively infects T cells, results in adaptation of the virus and production of a variant, NL4-3(M). Unlike NL4-3, NL4-3(M) has a rapid cytopathic effect in HL-60 and other myeloid cell lines. OBJECTIVE: To demonstrate that the tropism of NL4-3(M) is extended to primary monocyte-derived macrophages (MDM), and to determine whether the envelope gene, env, of NL4-3(M) is responsible for cytopathicity in HL-60 cells and replication in MDM. METHODS: A chimeric virus (NL4-3envA) containing the majority of env of NL4-3(M) was generated, and tested for virus replication and cytopathic effect in H9 and HL-60 cells, as well as for virus replication in primary MDM. To assess virus replication, the cultures were analyzed for expression of viral envelope glycoproteins on the infected cells and production of extracellular HIV-1 p24 antigen. Cytopathic effect on HL-60 cells was evaluated by monitoring the viabilities of the cultures. In addition, the majority of env of NL4-3envA was sequenced. RESULTS: The biological phenotypes of NL4-3, NL4-3(M), and NL4-3envA are distinctly different. Although both NL4-3(M) and NL4-3envA replicate in MDM, only NL4-3(M) is rapidly cytopathic in HL-60 cells. Nine amino-acid changes were identified within the envelope glycoproteins of NL4-3envA compared with NL4-3. CONCLUSIONS: The viral determinants of NL4-3(M) sufficient to extend the tropism of this virus to MDM reside, in part, in env. These genetic determinants are distinct from the viral determinants that control the cytopathic phenotype of this virus in HL-60 cells.

Binding Sites↗

Volumetric determination of placental and uterine growth relationships from B-mode ultrasound by serial area-volume determinations.

Serial area-volume determinations may be made from B-mode ultrasonography. Multiple sonographic sections are taken through a structure of interest. The area of the structure of interest is determined on each sonographic section by a form of planimetry and multiplied by the step-interval to obtain the volume of the structure of interest between sonographic sections. The total volume of the structure of interest is the sum of the individual volumes. The volume is computed from both transverse and longitudinal images and an average obtained for improved accuracy. Total intrauterine volume and placental volume were determined by the serial area-volume method in 63 normal patients and the results were analyzed. Placental and total intrauterine volumes were also determined for five cases of intrauterine growth retardation. Although there is some error in the serial area-volume method, the initial evaluation of five cases with intrauterine growth retardation suggests a greater precision and accuracy than volume estimates based on prolate ellipsoid geometry. Placental volume may provide an acceptable parameter to screen for intrauterine growth retardation.

Female↗

Identification of different antigenic determinants within the synthetic multichain co-polymer poly(Tyr,Glu)-polyAla--polyLys as recognized by the chicken. I. Significance of the polyAla--polyLys backbone and identification of poly-D-alanine as the antigenic determinant in backbone-directed responses.

The multichain co-polymer poly(Tyr,Glu)-polyAla--polyLys, designated (T,G)-A--L, is normally thought to present only the poly(Tyr,Glu) sequences, designated (T,G), as antigenic determinants. Evidence is presented indicating that at least two different determinants in the (T,G)-A--L antigen are recognized by chickens. Studies in a partly inbred high-responder chicken strain reveal two major determinant systems: the (T,G) and the A--L in the (T,G)-A--L antigen. For one serum the antigenic determinant of the A--L backbone is shown to be a poly-D-alanine.

Alanine↗

Unidirectional responses to Mls determinants in vivo. Polyclonal T-cell responses to a single common determinant of Mls in different efficiencies?

Polyclonal anti-Mls responses of peripheral (mature) and thymic (immature) lymphocytes were studied in vivo in terms of local host-versus-graft and graft-versus-host reactions. The level of stimulatory activity differed between the various types of Mls antigens, with Mlsa and Mlsd having the highest levels. Mlsc a lower level, and Mlsb the lowest level. The immunogenicity of Mlsa and Mlsd may not be identical, since Mlsd mice responded to the Mlsa determinant, while Mlsa mice did not react with the Mlsd determinants. This suggests that the anti-Mls response is unidirectional. The fact that Mlsb mice made tolerant at birth to Mlsd (Mlsa) antigens behave like Mlsd (Mlsa) mice when responding to Mls antigens supports this suggestion. Furthermore, thymus cells from the Mlsa-tolerized BALB/c (Mlsb) mice were unresponsive to both Mlsa and Mlsd antigens, while those made tolerant to Mlsd were responsive to Mlsa. These results indicate that the polyclonal response to the strongly immunogenic Mlsa,d antigens is unidirectional, and that the immunogenicity of Mlsa and Mlsd is not identical. Based on the results of tolerance experiments and data on T-cell clones, we suggest that the difference in immunogenicity between Mlsa,b,d antigens is due to different efficiencies of the responding T-cell populations, probably because of a quantitative difference in a single common antigenic determinant expressed in each Mls haplotype, which results in different levels of stimulation of T cells according to the avidity for the determinant.

Animals↗

Immunochemical studies of beef pancreas tryptophanyl-tRNA synthetase and its fragments. Determination of the number of antigenic determinants and a comparison with tryptophanyl- tRNA synthetases from other sources and with reverse transcriptase from avian myeloblastosis virus.

The immunoglobulin G (IgG) fraction of the antiserum from rabbits immunized with homogeneous beef pancreas tryptophanyl-tRNA synthetase inhibits the enzyme activity in the reactions of both tRNATrp aminoacylation and tryptophan activation. Fab fragments of IgG act in a similar way. Common antigenic determinants have been detected in tryptophanyl-tRNA synthetases from beef, pig, chicken and rat livers using pure antibodies against beef pancreas tryptophanyl-tRNA synthetase. This observation indicates the evolutional stability of certain structural features of tryptophanyl-tRNA synthetases. The interaction of antibodies with the fragments of beef tryptophanyl-tRNA synthetase produced by endogenous and tryptic proteolysis of the enzyme has been studied. On third of the antiserum antibodies interacting with the C-terminal fragment of the enzyme (Mr approximately equal to 40000) inhibits its activity whereas the antibodies to the N-terminal fragment (Mr approximately equal to 20000) have no effect on the enzyme activity. The immunochemical identity of the two synthetase fragments differing in their enzymatic activity supports the assumption that the loss of enzymatic activity of the tryptic fragment is caused by lack of a small peptide which is retained in case of endogenous proteolysis; probably the amino acid residues of this peptide participate in formation of active centre of tryptophanyl-tRNA synthetase. A radioimmunochemical method is described for determining the number of antigenic determinants. One molecule of tryptophanyl-tRNA synthetase was found to bind 9 (+/- 1) molecules of Fab fragments. Antibodies against tryptophanyl-tRNA snythetase from beef pancreas do not inhibit noticeably the activity of reverse transcriptase from avian myeloblastosis virus. No antigenic determinants in common have been detected in reverse transcriptase and tryptophanyl-tRNA synthetase by radioimmunochemical assays.

Amino Acyl-tRNA Synthetases↗

Three month study of changes in the cornea after computer-determined and conventionally-determined contact lens fitting.

The conventional rigid-lens fitting method uses fluorescein to assess the tear layer beneath a trial lens on the corneal surface. A more advanced technique, which uses a computer program to determine the back surface specifications of a contact lens from a pre-set tear layer thickness, has been studied. No significant difference was found in terms of corneal physiological changes between the conventionally determined contact lenses and computer-determined contact lenses after a three-month wearing period. The results suggest that there is little difference in the two methods of fitting. However, the computer determined method was found to be more efficient in terms of chair time, and also can aid inexperienced rigid lens practitioners to select the appropriate lens back surface curves.

Computer-Aided Design↗

Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors.

For the pathogenic yeast Candida albicans, secreted aspartyl proteinase (Sap) activity has been correlated with virulence. A family consisting of at least eight SAP genes can be drawn upon to produce Sap enzymatic activity. In this study, the levels of Sap1, Sap2, and Sap3 isoenzymes were monitored under a variety of growth conditions for several strains, including strain WO-1, which alternates between two switch phenotypes, white (W) and opaque (O). When cultured under proteinase-inducing conditions, most strains and W cells produce Sap2, while O cells produce Sap1, Sap2, and Sap3. Both W and O cells of strain WO-1 produce Saps in enriched and defined media that do not induce Saps from other strains. The specific Sap isoenzyme that is produced is determined by the cell type, while the level of Sap production is determined by environmental factors. The levels and temporal regulation of the SAP mRNAs as determined by Northern (RNA) analysis were consistent with Sap protein levels and with previous results. S1 analysis showed that SAP6 is the predominant SAP gene transcribed during hyphal induction at neutral pH. These studies define the culture conditions which control the levels of SAP mRNAs and Sap proteins, and they indicate that both the yeast/hyphal transition and phenotypic switching can determine which of the Sap isoenzymes is produced.

Aspartic Acid Endopeptidases↗

Multiple determinants contribute to the virulence of HSV ocular and CNS infection and identification of serine 34 of the US1 gene as an ocular disease determinant.

PURPOSE: The virulence of any given strain of herpes simplex virus (HSV) is probably due to the effects of the constellation of genes in that strain and how they act in concert to promote disease. The goal of this work was to develop a system to identify and study the role of multiple genes in HSV disease. METHODS: Mixed ocular infection with HSV-1 strains CJ394 and OD4 yield recombinants with increased ocular and central nervous system (CNS) virulence. Clones and subclones of the CJ394 genome were cotransfected with intact OD4 DNA into Vero cells, the transfection pools were inoculated into BALB/c mouse eyes, and disease severity was scored. Fragments transferring increased ocular or CNS disease were sequenced. Site-directed mutagenesis was used to revert one mutation to wild type. RESULTS: Five of the determinants (UL9, -33, -41, and -42 and US1) increased ocular disease when transferred singly. Transfer of the UL36/37 determinant increased both ocular and CNS disease. Transfer of the UL41 and -42 genes increased mortality and a combination of the UL36/37, -41, and -42 determinants increased virulence further. Reversion of the S34A change in the OD4 US1 gene to wild type restored ocular virulence. CONCLUSIONS: Multiple HSV genes can operate to increase virulence. The UL9, -33, -36/37, and -42 genes have not previously been identified as virulence determinants. The UL41 and US1 genes are known to affect disease, but the changes identified had not been described. Multiple novel mutations were found in the OD4, UL9, UL36, and US1 genes, and we showed that S34 in the US1 gene is essential in ocular disease.

Animals↗

Immunoturbidimetric determination of C-reactive protein (CRP) and high-sensitivity CRP on heparin plasma. Comparison with serum determination.

Determination of C-reactive protein (CRP) and high-sensitivity CRP (hs-CRP), markers of systemic inflammation and atherosclerosis risk, usually requires serum samples, while heparin plasma samples are routinely collected for routine chemistry. This study evaluates the use of heparin plasma samples for hs-CRP and CRP determinations using immunoturbidimetric methods on an Olympus system. As a result, analytical performance, including linearity, imprecision values, and determination from 185 clinical samples, are not affected by the type of sampling, validating the use of heparin plasma samples for CRP and hs-CRP determination with Olympus reagents.

Blood Chemical Analysis↗

Correlation between urinary oestrogen levels determined by haemagglutination inhibition reaction and serum oestradiol levels determined by radioimmunoassay.

Measurement of urinary or blood oestrogen is essential for a precise monitoring of follicular development in gonadotrophin therapy. Blood oestradiol levels are considered most accurately to reflect follicular maturation. Although radioimmunoassays (RIA) for oestradiol yielding rapid results are now available, they require qualified technicians and equipment for RIA. Recently, a simple, rapid method for determining urinary total oestrogen has been developed by Mochida Pharmaceutical Co. Ltd., Japan. To determine whether this kit may be applicable to monitoring gonadotrophin therapy, urinary total oestrogen concentrations measured by the kit were compared with serum oestradiol levels determined by RIA in patients who were treated with gonadotrophin. Urinary total oestrogen concentrations in samples obtained either at 07.00 h (first morning urine specimen) or at 09.00 h significantly correlated with serum oestradiol levels. Thus, urinary total oestrogen concentrations determined by the kit appear to reflect follicular development accurately, and are considered applicable to monitoring gonadotrophin therapy. Correction of the urinary total oestrogen concentrations by the creatinine level significantly improved the correlation between urinary total oestrogen values and serum oestradiol levels. Therefore, the primary total oestrogen values should be corrected by the creatinine level.

Chorionic Gonadotropin↗

Putative aromatase inhibitor induces male sex determination in a female unisexual lizard and in a turtle with temperature-dependent sex determination.

Treatment of developing embryos of two diverse species of reptiles with fadrozole (a potent and specific nonsteroidal inhibitor of aromatase activity in mammals) resulted in the induction of male sex determination. In the first experiment, males were produced in an all-female parthenogenic species of lizard (Cnemidophorus uniparens). In the second experiment, male sex determination was induced in a turtle (Trachemys scripta) with temperature-dependent sex determination. The results support the hypothesis that the endogenous production of oestrogen may represent a pivotal step in the sex determination cascade of reptiles. Further, the production of male C uniparens indicates that the genes required for male sexual differentiation have not been lost in this parthenogenic lizard.

Animals↗

Kinetic flow-injection determination of hydrogen peroxide by use of iron(III)-catalyzed coloration and its application to the determination of biological substances.

A kinetic flow-injection (FI) method is described for the determination of hydrogen peroxide. This method is based on an iron(III)-catalyzed oxidative coupling of 4-aminoantipyrine with N,N-dimethylaniline by hydrogen peroxide. By measuring the change in the absorbance of the dye formed at 560 nm, 1 x 10(-6) - 6 x 10(-4) M hydrogen peroxide could be determined with a sampling rate of 15 h(-1). The relative standard deviation (n = 30) was 0.8% for 5 x 10(-5) M hydrogen peroxide. There was little interference of the co-existing ions and compounds. After introducing some immobilized enzyme reactors to the FI system, the proposed method allowed the determination of glucose and uric acid ranging from 1 x 10(-6) to 6 x 10(-4) M with relative standard deviations of below 2%. The applicability of the method was demonstrated by determining these substances in serum samples.

Ampyrone↗

Cutting edge: introduction of an endopeptidase cleavage motif into a determinant flanking region of hen egg lysozyme results in enhanced T cell determinant display.

The choice of which determinants of a whole Ag will be presented on cell surface MHC class II molecules after uptake and processing by APC is the result of the interplay between structural characteristics of the Ag and the processing machinery of the APC. In this study, we demonstrate that introduction of a dibasic motif adjacent to a subdominant determinant enhances the presentation of this determinant from the whole molecule. This is the first report showing that a single amino acid substitution in a whole Ag, designed to introduce an endopeptidase recognition site, enhances display of class II-restricted determinants, most likely by creating a peptide chain cleavage in the antigenic molecule. Our findings have important implications for the understanding of immunodominance and for vaccine design.

Amino Acid Motifs↗

Determination of phytic acid in cereals using ICP-AES to determine phosphorus.

A novel approach for determination of phytic acid in cereals has been applied in 2 traditional methods. In the first, phytic acid in a sample extract is first separated and concentrated by ion-exchange chromatography. The phytic acid concentrate is then quantitatively determined as phosphorus by inductively coupled plasma atomic emission spectrometry (ICP-AES). In the second method, extracted phytic acid is first precipitated by FeCl3 solution. The complexed iron is converted to ferric hydroxide by adding NaOH, thus releasing phytic acid as soluble sodium phytate. Phytate is then quantitatively determined as phosphorus by ICP-AES. In these methods, both the difficult acid digestion and the spectrometric determination of phosphorus found in traditional methods are eliminated by using ICP-AES. This results in a method that is simpler, faster, and more accurate than earlier procedures.

Chromatography, Ion Exchange↗

Combination of total solids determined by oven drying and fat determined by Mojonnier extraction for measurement of solids-not-fat content of raw milk: collaborative study.

Each of 9 laboratories analyzed 9 pairs of blind duplicate raw milk samples for fat, using the Mojonnier ether extraction method (16.E13-16.E17), and for total solids, using a new direct forced air oven method. Solids-not-fat was determined by subtracting percent fat from percent total solids. The solids-not-fat content of the samples tested in the collaborative study ranged from 8.48 to 9.36%. The average repeatability standard deviation (sr) and the average reproducibility standard deviation (SR) for the solids-not-fat method were 0.019 and 0.041, respectively. Average repeatability (RSDr) and reproducibility (RSDR) relative standard deviations were 0.218 and 0.466%, respectively. The mean repeatability value (r) was 0.055; the mean reproducibility value (R) was 0.117. The difference between milk total solids determined by direct forced air oven drying and milk fat determined by Mojonnier ether extraction has been approved for interim official first action for determination of solids-not-fat content of milk.

Animals↗

IgD allotypic determinants. II. Determinants expressed on IgD of mice of the b, e, and o Igh-C haplotypes.

Allotypic determinants expressed on membrane IgD of 27 strains of mice representing each of the major Igh-C haplotypes of inbred mice were examined with reagents specific for the IgD of mice of the b, e, and o Igh-C haplotypes. The distribution of two specificities, IgD.44 (Ig5.2) and IgD.37 (Ig5.3), recognized by two monoclonal anti-delta reagents was determined. In addition, a new specificity, IgD.46, expressed on mice of the o Igh-C type, but not of the b or a types, was detected by a BALB/c anti-AL/N spleen cell antiserum. The results also confirmed the description of the new Igh-C haplotype o, by demonstrating that the mice of this group express the IgD.37 determinant. This determinant is also expressed by mice of the b and e types, but not by mice of the d haplotype, from which the o haplotype was subdivided.

Animals↗