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Results for “second generation sequencing”

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PubMed2005

Levalbuterol versus racemic albuterol in the treatment of acute exacerbation of asthma in children.

OBJECTIVE: To compare levalbuterol and racemic albuterol for the treatment of acute exacerbation of asthma in pediatric population. DESIGN: Prospective, double-blind, randomized research trial in a pediatric emergency department of an urban tertiary care hospital. PARTICIPANTS: Children 5 to 21 years with a history of asthma presenting to the emergency department in acute exacerbation. INTERVENTIONS: As per a computer-generated randomization sequence, patients received either 1.25 mg of levalbuterol or albuterol 2.5 mg via nebulization along with ipratropium hydrochloride. Patients received 3 back-to-back treatments as needed every 20 minutes, maximum of 3; 2 mg/kg of oral prednisone was administered to the patients after the second treatment. Baseline respiratory parameters such as oxygen saturations, respiratory rates, and peak flow rates were measured and repeated after every treatment.The decision for further treatments and or hospitalization was made by the treating emergency department physician as per his/her clinical judgement of the respiratory parameters at the end of 3 treatments. RESULTS: Seventy patients completed the study. Most of the patients were in moderate severity of asthma exacerbation. All patients in both groups showed improvement in oxygen saturations, respiratory rates, and peak flow rates. However, no statistically significant difference was observed in the 2 groups regarding the respiratory parameters (P > 0.05). CONCLUSION: Levalbuterol is not more efficacious than racemic albuterol in improving respiratory parameters in children presenting with acute exacerbation of asthma.

Adolescent↗

PubMed2000

A segmental gene duplication generated differentially expressed myb-homologous genes in maize.

The myb-homologous p1 gene regulates the synthesis of flavonoid pigments in maize kernel pericarp and cob; these floral organs are greatly modified in size and shape compared with their counterparts in teosinte, the progenitor of maize. To elucidate the molecular evolution of the p1 gene in relation to its expression and possible functions in maize and teosinte, we have isolated a second maize gene (p2) that is highly homologous with the p1 gene, and a related gene (p2-t) from Zea mays subsp parviglumis. We present evidence that the maize p1 and p2 genes were generated by duplication of an ancestral p gene (p(pre)) and its downstream sequences; the duplicated 3' flanking sequences were inserted upstream of the p(pre) gene, thereby changing its transcription pattern. This model accounts for the structural organization and the observed differential expression of the p1 and p2 genes: p1 transcripts accumulate in kernel pericarp, cob, tassel glumes, and silk, whereas p2 transcripts are found in developing anther and silk. The duplication is estimated to have occurred 2.75 million years ago; subsequently, multiple retroelements have been inserted between the p1 and p2 genes. Our results demonstrate the evolution of a single gene into a compound locus containing two component genes with different tissue specificities. Expression of the p1 gene in the kernel pericarp may have provided a selective advantage during the evolution of maize kernel morphology.

Amino Acid Sequence↗

PubMed2004

Identification of humans using gait.

We propose a view-based approach to recognize humans from their gait. Two different image features have been considered: the width of the outer contour of the binarized silhouette of the walking person and the entire binary silhouette itself. To obtain the observation vector from the image features, we employ two different methods. In the first method, referred to as the indirect approach, the high-dimensional image feature is transformed to a lower dimensional space by generating what we call the frame to exemplar (FED) distance. The FED vector captures both structural and dynamic traits of each individual. For compact and effective gait representation and recognition, the gait information in the FED vector sequences is captured in a hidden Markov model (HMM). In the second method, referred to as the direct approach, we work with the feature vector directly (as opposed to computing the FED) and train an HMM. We estimate the HMM parameters (specifically the observation probability B) based on the distance between the exemplars and the image features. In this way, we avoid learning high-dimensional probability density functions. The statistical nature of the HMM lends overall robustness to representation and recognition. The performance of the methods is illustrated using several databases.

Algorithms↗

PubMed1993

Second-generation hepatitis C Elisa antibody tests confirmed by the four-antigen recombinant immunoblot assay correlate well with hepatitis C viremia and chronic liver disease in Swedish blood donors.

Seventy-three Swedish blood donors (52 men, 21 women; median age 36 years) repeatedly reactive for hepatitis C antibodies (anti-HCV C-100-3) were tested with a second-generation (2nd-gen) anti-HCV Elisa and a 4-band recombinant immunoblot assay (RIBA 2). These results were correlated to serum alanine aminotransferase (S-ALAT), liver morphology and viremia as detected by 'nested' polymerase chain reaction (PCR) based on primers from a 5'-noncoding sequence of the HCV genome. Thirty-five of 46 (76%) donors with positive 2nd-gen Elisa tests confirmed by RIBA 2 were PCR positive whereof 27 had histological findings compatible with chronic persistent hepatitis (CPH) and 7 had chronic active hepatitis (CAH). Ten of 56 (18%) 2nd-gen Elisa-positive donors were RIBA 2 negative (or indeterminate) and none of these had chronic hepatitis nor were PCR positive. Seventeen of 73 (23%) donors were 1st-gen Elisa positive but 2nd-gen Elisa negative. All of these were PCR negative and only 1 (6%) had chronic hepatitis (CPH). An elevated S-ALAT level (reference < 0.7 mu kat/l) was found in 26 2nd-gen Elisa and RIBA 2-positive donors of which 18 had CPH and 7 had CAH and all 25 were PCR positive. A normal S-ALAT level was found in 9 of 34 (26%) donors with chronic hepatitis (all had CPH) and positive PCR. We have found that blood donors with positive 2nd-gen anti-HCV Elisa tests confirmed by RIBA-2 and especially with a concomitant elevated S-ALAT are highly likely to be viremic as demonstrated by PCR and to have chronic hepatitis.

Adult↗

PubMed1994

Genuine and apparent cross-reaction of polyclonal antibodies to proteins and peptides.

Antiserum to a native protein may cross-react with the corresponding denatured protein or with peptides. The cross-reaction is either a genuine property of the antibodies or caused by antibodies produced against some unfolded protein contaminating the native protein used for immunization. Appropriate conformation-sensitive immunoassays must be employed to distinguish a genuine from an apparent cross-reaction. In the present study, we have analyzed critically the cross-reaction of rabbit antisera against proteins and peptides. We have distinguished between genuine and apparent cross-reaction with the help of the protein A antibody-capture ELISA, a new conformation-sensitive ELISA format. Three systems were analyzed: cross-reaction of antisera to native yeast and horse cytochrome c with unfolded apo-cytochrome c; cross-reaction of antisera to a coiled-coil leucine-zipper peptide with a homologous random-coil peptide obtained by introducing two proline residues into the leucine-zipper sequence; cross-reaction of antisera to two peptides that correspond to the N-terminal and an internal sequence of ferredoxin: NADP+ reductase (FNR), with the native enzyme. The reaction of the anti-(cytochrome c) sera was clearly due to antibodies produced against unfolded protein, it was an apparent and not a genuine cross-reaction. Furthermore, the apparently cross-reactive antibodies to horse cytochrome c did not discriminate against sequence-related proteins from dog, beef, rabbit and pigeon. In contrast, antibodies to the leucine-zipper peptide did cross-react in a genuine way with the homologous random-coil peptide, that is, the cross-reactive antibodies do not seem to have been produced against the unfolded form of the leucine-zipper peptide. Of the two anti-peptide sera the one against the unstructured and highly accessible N-terminal segment reacted strongly with the native protein. The second serum against a solvent-accessible turn-like sequence of FNR showed apparent cross-reactivity: antibodies recognizing the native protein were directed against a minor conformational isoform of the free peptide and did not react with the principal form(s) of the free peptide. The generation of cross-reactive antibodies depends on the conformational stability and integrity of the immunogen and on the molecular form of its application, i.e., free, polymerized or carrier-bound. The results clarify the different nature of cross-reactivity of antisera to proteins and peptides. This knowledge is crucial if antisera are to be used as conformation-specific probes.

Amino Acid Sequence↗

PubMed1999

Identification of rhythmically active cells in guinea-pig stomach.

1. When intracellular recordings were made from the antral region of guinea-pig stomach, cells with different patterns of electrical activity were detected. 2. One group of cells, slow-wave cells, generated slow waves which consisted of initial and secondary components. When filled with either Lucifer Yellow or neurobiotin, the cells identified as smooth muscle cells lying in the circular muscle layer. 3. A second group of cells, driving cells, generated large, rapidly rising, potential changes, driving potentials. They had small cell bodies with several processes. With neurobiotin, a network of cells was visualized that resembled c-kit positive interstitial cells of the myenteric region. 4. A third group of cells generated sequences of potential changes which resembled driving potentials but had smaller amplitudes and slow rates of rise. These cells resembled smooth muscle cells lying in the longitudinal muscle layer. 5. When simultaneous recordings were made from the driving and slow-wave cells, driving potentials and slow waves occurred synchronously. Current injections indicated that both cell types were part of a common electrical syncytium. 6. The initial component of slow waves persisted in low concentrations of caffeine, but the secondary component was abolished; higher concentrations shortened the duration of the residual initial component. Driving potentials continued in the presence of low concentrations of caffeine; moderate concentrations of caffeine shortened their duration. 7. Hence three different types of cells were distinguished on the basis of their electrical activity, their responses to caffeine and their structure. These were smooth muscle cells, lying in the longitudinal and circular layers, and interstitial cells in the myenteric region. The observations suggest that interstitial cells initiate slow waves.

Animals↗

PubMed1992

Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors.

The pathogenic Neisseria species are capable of utilizing transferrin as their sole source of iron. A neisserial transferrin receptor has been identified and its characteristics defined; however, the biochemical identities of proteins which are required for transferrin receptor function have not yet been determined. We identified two iron-repressible transferrin-binding proteins in Neisseria gonorrhoeae, TBP1 and TBP2. Two approaches were taken to clone genes required for gonococcal transferrin receptor function. First, polyclonal antiserum raised against TBP1 was used to identify clones expressing TBP1 epitopes. Second, a wild-type gene copy was cloned that repaired the defect in a transferrin receptor function (trf) mutant. The clones obtained by these two approaches were shown to overlap by DNA sequencing. Transposon mutagenesis of both clones and recombination of mutagenized fragments into the gonococcal chromosome generated mutants that showed reduced binding of transferrin to whole cells and that were incapable of growth on transferrin. No TBP1 was produced in these mutants, but TBP2 expression was normal. The DNA sequence of the gene encoding gonococcal TBP1 (tbpA) predicted a protein sequence homologous to the Escherichia coli and Pseudomonas putida TonB-dependent outer membrane receptors. Thus, both the function and the predicted protein sequence of TBP1 were consistent with this protein serving as a transferrin receptor.

Amino Acid Sequence↗

PubMed1994

The Bel1 protein of human foamy virus contains one positive and two negative control regions which regulate a distinct activation domain of 30 amino acids.

The Bel1 transactivator is essential for the replication of human foamy virus (HFV). To define the functional domains of HFV Bel1, we generated random missense mutations throughout the entire coding sequence of Bel1. Functional analyses of 24 missense mutations have revealed the presence of at least two functional domains in Bel1. One domain corresponds to a basic amino acid-rich motif which acts as a bipartite nuclear targeting sequence. A second, central domain corresponds to a presumed effector region which, when mutated, leads to dominant-negative mutants and/or lacks transactivating ability. In addition, deletion analyses and domain-swapping experiments further showed that Bel1 protein contains a strong carboxy-terminal activation domain. The activating region is also capable of functioning as a transcription-activating domain in yeast cells, although it does not bear any significant sequence homology to the well-characterized acidic activation domain which is known to function only in yeast and mammalian cells. We also demonstrated that the regions of Bel1 from residues 1 to 76 and from residues 153 to 225 repressed transcriptional activation exerted by the Bel1 activation domain. In contrast, the region from residues 82 to 150 appears to overcome an inhibitory effect. These results indicate that Bel1 contains one positive and two negative regulatory domains that modulate a distinct activation domain of Bel1. These regulatory domains of Bel1 cannot affect the function of the VP16 activation domain, suggesting that these domains specifically regulate the activation domain of Bel1. Furthermore, in vivo competition experiments showed that the positive regulatory domain acts in trans. Thus, our results demonstrate that Bel1-mediated transactivation appears to undergo a complex regulatory pathway which provides a novel mode of regulation for a transcriptional activation domain.

Amino Acid Sequence↗

PubMed2006

Ablation of undifferentiated human embryonic stem cells: exploiting innate immunity against the Gal alpha1-3Galbeta1-4GlcNAc-R (alpha-Gal) epitope.

Although undifferentiated human embryonic stem cells (hESCs) are tumorigenic, this capacity is lost after differentiation, and hESCs are being widely investigated for applications in regenerative medicine. To engineer protection against the unintentional transplantation of undifferentiated cells, we generated hESCs carrying a construct in which the alpha1,3-galactosyltransferase (GalT) open reading frame was transcribed from the hTERT promoter (pmGT). Because the endogenous GalT gene is inactive, GalT expression was limited to undifferentiated cells. A second chimeric construct (pmfGT) differed by replacement of the GalT leader sequence for that of the fucosyltransferase gene. Two subclones containing stable integrations of pmGT and pmfGT (M2 and F11, respectively) were assessed for their response to human serum containing antibodies to the Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal) epitope. The low-variegation line, M2, and to a lesser extent the more variegated line F11, were sensitive to human serum when exposed in the undifferentiated state. However, M2 cells were largely insensitive after differentiation and retained both a normal karyotype and the ability to differentiate into derivatives of the three germ layers in severe combined immunodeficient mice. These data exemplify a method of protection against residual, undifferentiated hESCs prior to engraftment and may provide ongoing immune surveillance after engraftment against dedifferentiation or against de novo tumorigenesis involving hTERT reactivation. Untransfected H9 cells were not sensitive to the human serum used in this study. Hence, in our system, interactions of hESCs with other circulating antibodies, such as anti-Neu5Gc, were not observed.

ABO Blood-Group System↗

PubMed2005

Transcriptional regulation of the human CD3 gamma gene: the TATA-less CD3 gamma promoter functions via an initiator and contiguous Sp-binding elements.

Growing evidence that the CD3gamma gene is specifically targeted in some T cell diseases focused our attention on the need to identify and characterize the elusive elements involved in CD3gamma transcriptional control. In this study, we show that while the human CD3gamma and CD3delta genes are oriented head-to-head and separated by only 1.6 kb, the CD3gamma gene is transcribed from an independent but weak, lymphoid-specific TATA-less proximal promoter. Using RNA ligase-mediated rapid amplification of cDNA ends, we demonstrate that a cluster of transcription initiation sites is present in the vicinity of the primary core promoter, and the major start site is situated in a classical initiator sequence. A GT box immediately upstream of the initiator binds Sp family proteins and the general transcription machinery, with the activity of these adjacent elements enhanced by the presence of a second GC box 10 nt further upstream. The primary core promoter is limited to a sequence that extends upstream to -15 and contains the initiator and GT box. An identical GT box located approximately 50 nt from the initiator functions as a weak secondary core promoter and likely generates transcripts originating upstream from the +1. Finally, we show that two previously identified NFAT motifs in the proximal promoter positively (NFATgamma(1)) or negatively (NFATgamma(1) and NFATgamma(2)) regulate expression of the human CD3gamma gene by their differential binding of NFATc1 plus NF-kappaB p50 or NFATc2 containing complexes, respectively. These data elucidate some of the mechanisms controlling expression of the CD3gamma gene as a step toward furthering our understanding of how its transcription is targeted in human disease.

Base Sequence↗

PubMed1990

Importance of subtype in the immune response to the pre-S(2) region of the hepatitis B surface antigen. II. Synthetic Pre-S(2) immunogen.

One purpose of this study was to examine the influence of viral subtype on in vivo antibody production to the pre-S(2) region of the hepatitis B surface Ag. Immunization with hepatitis B surface Ag particles containing the pre-S(2) region of the d or y subtypes identified the B10.M (H-2f) strain as an antibody nonresponder to the pre-S(2) and S regions after immunization with the y subtype, but as an antibody responder to the pre-S(2) and S regions after immunization with the d subtype. Both the S region and pre-S(2) region-specific antibody responses emanated from pre-S(2)/d-specific Th cell function because B10.M mice are T cell nonresponsive to the S region of both subtypes. Although responder/nonresponder status of the B10.M strain was dependent on the pre-S(2) subtype used for immunization, the anti-pre-S(2) antibody produced was totally cross-reactive on both subtypes. This is consistent with the conserved nature of the dominant pre-S(2) antibody-binding site and the highly polymorphic nature of the pre-S(2) sequence that represents the focus of T cell recognition. These data suggest that, to fully benefit from the inclusion of pre-S(2) region sequences, third generation hepatitis B virus vaccines should contain both the d and y subtype sequences of the pre-S(2) region to increase the frequency of pre-S(2) and S-specific antibody responses and to insure Th cell memory relevant to both viral subtypes. A second purpose of this study was to "design" a synthetic pre-S(2) immunogen based on combining the dominant B and T cell recognition sites into a single peptide. A composite peptide consisting of the dominant T cell recognition sequence p151-174 positioned N-terminal to the dominant B cell site p133-143 (i.e., p151-174(133-143] yielded an effective pre-S(2) synthetic immunogen. Interestingly, the orientation of the T and B cell determinants and the context of the T cell site within the larger composite peptide influenced both antibody fine specificity and T cell fine specificity.

Amino Acid Sequence↗

PubMed1993

Cloning and characterization of rabbit liver UDP-glucuronosyltransferase cDNAs. Developmental and inducible expression of 4-hydroxybiphenyl UGT2B13.

A polyclonal antibody generated against rabbit liver p-nitrophenol UDP-glucuronosyltransferase (UGT) was used to screen a rabbit liver cDNA expression library constructed in lambda gt11. A 500-base pair cDNA clone, termed pPNP, generated a fusion protein that was antigenic with the antibody. Clone pPNP encoded the 3' region of a UGT. To identify larger recombinants, clone pPNP was used as a probe to screen a second cDNA library constructed in lambda ZAP. Two different cDNA clones were identified by DNA sequence analysis. Based upon their predicted amino acid sequence analysis, the clones encode transferases belonging to the UGT2 subfamily, and have been identified as UGT2B13 and UGT2B14. The predicted N-terminal sequence of UGT2B13 is identical to that determined for the purified rabbit liver estrone UGT. However, expression of the UGT2B13 cDNA in COS-1 cells displayed no activity in the presence of estrone but efficiently conjugated 4-hydroxybiphenyl. Results of Southern blot analysis using the 5' divergent region of the UGT2B13 cDNA that encodes exon 1 demonstrates that multiple genes share sequence homology to UGT2B13, an observation which indicates that the estrone UGT and UGT2B13 genes are encoded by separate alleles. When the 5' variable regions of the cDNAs where used in Northern blot analysis, the expression of UGT2B13 and UGT2B14 were shown to be expressed primarily in adult rabbits. However, when neonatal rabbits were treated with either dexamethasone or rifampicin, UGT2B13 mRNA levels were induced. The neonatal induction of UGT2B13 mRNA corresponded with similar increases in 4-hydroxybiphenyl UGT activity. The expression and induction of UGT2B13 paralleled that of the developmentally regulated rabbit liver progesterone 6 beta-hydroxylase P4503A6.

Amino Acid Sequence↗

PubMed1998

Dominantly transmitted beta-thalassemia arising from the production of several aberrant mRNA species and one abnormal peptide.

We describe a dominantly inherited beta-thalassemia intermedia phenotype observed in a five-generation Portuguese family. Carriers are characterized by moderate anemia, hypochromia, microcytosis, elevated hemoglobin (Hb)A2 and HbF levels, splenomegaly, hepatomegaly, and inclusion bodies in peripheral red blood cells after splenectomy. The molecular basis of this condition is a small deletion within the 5' consensus splicing sequence of the second intron of the beta-globin gene, IVS-II-4,5 (-AG). Reticulocyte RNA studies performed by reverse transcription-polymerase chain reaction (RT-PCR) and primer extension analysis showed three abnormally processed transcripts, which, upon sequencing, were shown to correspond to (1) skipping of exon 2, and (2) activation of two cryptic splice sites (between codons 59/60, and at IVS-II-47). In vitro translation studies of these patients' reticulocyte RNA have shown that at least one of these aberrant mRNA species is translated into an abnormally elongated peptide whose cytotoxic properties could, in part, be causing the atypical dominant mode of inheritance observed in this family. We suggest that this elongated beta chain is unable to combine with an alpha-globin chain to form a functional Hb molecule. Its degradation would, then, exhaust the proteolytic defense mechanism of the erythroid precursors, leading to inefficient proteolysis of the free alpha chains in excess.

Amino Acid Sequence↗

PubMed2001

Characterization and crystallization of a novel Sarcocystis muris lectin, SML-2.

A novel lectin (SML-2) consisting of 138 amino acids was isolated from cyst merozoites of Sarcocystis muris and sequenced by Edman degradation and mass spectrometry. All 12 cysteinyl residues are involved in disulfide bridges, four of which are attributed to a characteristic pattern of cysteines as found in the so-called PAN-module superfamily. Crystals of SML-2 diffracting to 2.1 A resolution at a synchrotron were grown by the hanging-drop vapour-diffusion technique. They belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 53.6, b = 128.8, c = 158.2 A and eight molecules in the asymmetric unit. SML-2 cocrystallized with Au galactose results in two different crystal forms. The first form is isomorphous with the native crystals and the second form adopts space group C222(1), with unit-cell parameters a = 74.7, b = 82.0, c = 131.0 A, and diffracts to 2.4 A at a rotating-anode X-ray generator.

Amino Acid Sequence↗

PubMed1988

Rift Valley fever virus M segment: cell-free transcription and translation of virus-complementary RNA.

A cell-free system has been used to study gene expression of the M segment RNA of the Phlebovirus Rift Valley fever virus (RVFV). RVFV sequence-containing plasmids were used to synthesize M segment mRNA-like transcripts. These transcripts were then translated in vitro in the absence or presence of microsomal membranes. Cell-free translation of a transcript which closely resembled authentic M segment mRNA (RNA-7) yielded a primary translation product of 133 kilodaltons (kDa), the size expected of a polypeptide encompassing the entire open reading frame (ORF) of the M segment. When translations were conducted in the presence of microsomal membranes, this primary protein was cotranslationally processed to yield the two viral glycoproteins, G1 and G2, as well as proteins of 78, 21, and 14 kDa. With one exception, these in vitro processed polypeptides comigrated with M segment-encoded proteins found in RVFV-infected cell lysates. A polypeptide corresponding to the in vitro 21-kDa protein was not detected in vivo. To investigate translational initiation and processing of the protein products of the M segment, additional transcripts were generated in which varying portions of the amino-terminal "preglycoprotein" region of the M segment ORF were deleted. Translation results indicated that the 78- and 21-kDa proteins were initiated from the first methionine codon of the ORF, and the 14-kDa polypeptide began from the second in-phase ATG. These products and a major portion of the preglycoprotein region sequence were not required for the proper synthesis and processing of the viral glycoproteins in vitro. In light of these results, possible expression strategies used by this Phlebovirus M segment RNA are discussed.

Bunyaviridae↗

PubMed1996

Analysis of a gene that suppresses the morphological defect of bald mutants of Streptomyces griseus.

When present in multiple copies, orf1590 restored sporulation to class IIIA bald mutants of Streptomyces griseus, which form sporulation septa and thick spore walls prematurely. The orf1590 alleles from class IIIA bald mutants restored sporulation upon introduction at a high copy number into those same mutants, and the nucleotide sequence of one of these alleles was identical to that of the wild-type strain. We conclude that overexpression of orf1590 suppresses the defect in class IIIA bald mutants. Previous nucleotide sequence and transcript analyses suggested that orf1590 could encode two related proteins, P56 and P49.5, from nested coding sequences. A mutation that prevented the synthesis of P56 without altering the coding sequence for P49.5 eliminated the function of orf1590, as did amino acid substitutions in the putative helix-turn-helix domain located at the N terminus of P56 and absent from P49.5. To determine the coding capacity of orf1590, we analyzed translational fusions between orf1590 and the neo gene from Tn5. Measurement of the expression of fusions to the wild-type and mutant alleles of orf1590 indicated that P56 was the sole product of orf1590 during vegetative growth. Attempts to generate a nonfunctional frameshift mutation in orf1590 were unsuccessful in the absence of a second-site bald mutation, suggesting that orf1590 may be required during vegetative growth by preventing early sporulation. Our results are consistent with the hypothesis that P56 at a high level delays the premature synthesis of sporulation septa and spore walls in class IIIA mutants.

Amino Acid Sequence↗

PubMed1987

Cell-dependent chemiluminescence. Modulation of the N-formyl chemotactic peptide (FNLPNTL) mediated oxidative burst in human polymorphonuclear leukocytes (PMNL) by murine monoclonal antibody NMS-1.

Binding of purified monoclonal antibody (moAB) IgM NMS-1 to suspended initially spherical living human PMNLs is not associated with the generation of chemiluminescence but was found to enhance the chemiluminescence response to the N-formyl chemotactic peptide FNLPNTL. We investigated quantitatively the kinetics of oxygen metabolite generation by PMNLs stimulated with FNLPNTL +/- moAB NMS-1 using luminol-dependent chemiluminescence as a very sensitive detection system. Chemiluminescence detection allowed the analysis of the time sequence of onset and development of reactive oxygen metabolites following stimulation of PMNLs by FNLPNTL in the presence of moAB NMS-1. The increase of response of PMNLs stimulated with FNLPNTL in the presence of moAB NMS-1 depended on the concentration of the antibody and the sequence of stimulus addition. Stimulation of human PMNLs by 10 nM FNLPNTL induced a rapid burst of chemiluminescence which peaked approximately 5 min after stimulus addition. The subsequent addition of moAB NMS-1 (greater than or equal to 2 micrograms/ml DPBS(+) - 0.1% HSA, 37 degrees C) to FNLPNTL-stimulated PMNLs - after the FNLPNTL-mediated response had already decayed (16-18 min) - without delay induced a second burst of oxygen metabolite generation. The magnitude of this second peak of activation was dose-dependent. Treatment of PMNLs with moAB NMS-1 (greater than or equal to 1 microgram/ml DPBS(+) - 0.1% HSA, 3 min, 37 degrees C) - prior to FNLPNTL (10 nM) stimulation - increased rate and magnitude of the FNLPNTL-mediated response. This response is biphasic with the first peak at the FNLPNTL position and a second, higher peak approximately 16 min after FNLPNTL addition. The magnitude of response was dose-dependent. The latency (lag time) of the response was not changed compared to controls which received no moAB NMS-1 treatment. The observed moAB NMS-1 dependent increase in FNLPNTL-mediated chemiluminescence is transient (50-60 min), persistent activation was not detected.

Antibodies, Monoclonal↗

PubMed1999

Meta-PCR: a novel method for creating chimeric DNA molecules and increasing the productivity of mutation scanning techniques.

Many mutation scanning techniques are capable of locating mutations in DNA fragments much larger than the average exon. We have developed a system called Meta-PCR that can maximize the length of sequence scanned by these techniques, improving their productivity and realizing their full potential. Meta-PCR is a simple, versatile, and powerful method for generating chimeric DNA molecules. Currently, up to five PCR amplifiable fragments can be combined to form a single linear amplimer. The Meta-PCR reaction is self-assembling and takes place in two coupled stages carried out in a single reaction vessel. The order of fragments is reproducible and determined by primer design. We have developed two Meta-PCR assays, one comprising exons 6-10 of the Neurofibromatosis type 2 (NF2) gene and the second exons 8-12 of the human mismatch repair gene, hMLH1. We verified by direct sequencing that the order and sequence of the component exons in the Meta-PCR products is as predicted. Meta-PCR products from seven previously ascertained heterozygotes for NF2 mutations were directly sequenced. All seven mutations were clearly visible as mixed bases at the expected nucleotide, confirming that Meta-PCR faithfully reproduces the original sample genotype. We have evaluated the downstream use of the NF2 Meta-PCR products in fluorescent solid-phase chemical cleavage of mismatches (CCM). Meta-PCR products from eleven NF2 mutant heterozygotes were screened retrospectively for piperidine cleavage after hydroxylamine or potassium permanganate modification of mismatched bases. Ten of the 11 mutants were detected by visible cleavage. One mutation predicted to be cleaved after potassium permanganate modification was not detected. However, we were able to attribute this false negative to a failure in the CCM method. Meta-PCR is likely to be useful to clinical molecular diagnostic laboratories, helping them to fulfill demand for rapid and accurate screening for point mutations in large multi-exon genes.

Adaptor Proteins, Signal Transducing↗