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PubMed1999

Characterization of kinetoplast DNA minicircles of an Indian isolate of Leishmania donovani.

Characterization of the kinetoplast DNA minicircles in a human pathogenic Indian isolate of Leishmania donovani has not been reported previously. Using inverse PCR, we constructed a library of PCR-amplified minicircle variable region from the kinetoplast DNA of this isolate. A combination of restriction enzyme digestion and nucleotide sequence analysis revealed five minicircle DNA sequence classes within the library, one of which was predominant, representing 75% of the kDNA network. Another distinct sequence class represented 15% of the minicircle network. Other minor sequence classes collectively constituted the remaining 10% of the network. Apart from generating basic information on the organisation and distribution of the different sequence classes within the minicircles, the DNA sequence analysis also revealed unique attributes to our minicircles. One was the surprising homology of our isolate (an Old World sp.) with distantly related New World Leishmania species. Secondly, open reading frames were also identified, indicating the possibility that these minicircles may have more than a structural role to play within the kinetoplast network.

Amino Acid Sequence↗

PubMed2000

Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants.

Transgenic plants expressing recombinant proteins from pathogenic microorganisms provide an inexpensive edible vaccine for induction of local immunity. Three transgenic plant lines were generated expressing the spike (S) protein of transmissible gastroenteritis virus (TGEV), a protein crucial for establishing mucosal immunity. All three of them were driven by a strong plant promoter. One construct contained the 3.7 kb 5' end of the native S gene sequence. In the second construct part of the S gene, from nucleotide 49 to 1785, was modified for optimal plant recognition and was fused to a plant signal peptide coding sequence. The third construct contained the D epitope-coding region of the S gene, from nucleotide 1201 to 1591, which was fused to the alfalfa beta-amylase gene. The S gene products were detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Antigens from all three transgenic plant lines induced TGEV-specific immune responses in pigs as determined by virus neutralization and ELISA, and the resultant antibody titers for all three constructs were similar.

Animals↗

PubMed1996

Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients.

Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised individuals and those with cystic fibrosis (CF). In CF patients, the secretion of abnormally high amounts of mucus into the airways contributes to their susceptibility to infection by P. aeruginosa. To identify virulence genes of P. aeruginosa that are important in infection of CF patients, an in vivo selection system (IVET) was used to identify promoters that are specifically inducible by respiratory mucus derived from CF patients. Three genetic loci that are highly inducible by the mucus were identified. One of them is a well-characterized virulence gene (fptA), encoding the receptor for pyochelin, which is a P. aeruginosa iron siderophore. Induction of the fptA gene by mucus is suppressed by the addition of exogenous iron, demonstrating that the mucus is an iron chelator and generates an iron-deficient environment in CF lungs. Therefore, as a part of the host-defence mechanism, the mucus could also be responsible for induction of iron-regulated virulence factors of bacterial pathogens. The second locus, np20, encodes a peptide that shares sequence homology to a number of transcriptional regulators. An identical locus was previously identified to be inducible in vivo during infection of mice and was shown to be important in bacterial virulence in a neutropenic-mouse infection model. The third locus, designated migA (mucus inducible gene), was sequenced and found to encode a 299-amino-acid peptide which is homologous to glycosyltransferases of other bacteria, and is involved in the biosynthesis of lipopolysaccharides or exopolysaccharides. Inducibilities of the np20 and migA genes are not affected by iron and the exact nature of the inducing signals in the mucus is not known. The possible implications of the migA inducibility by respiratory mucus is discussed in relation to the P. aeruginosa infection in CF.

Amino Acid Sequence↗

PubMed1988

Modulation of matrix adhesive responses of human neuroblastoma cells by neighboring sequences in the fibronectins.

Attachment and neurite extension have been measured when Platt or La-N1 human neuroblastoma cells respond to tissue culture substrata coated with a panel of complementary fragments from the individual chains of human plasma (pFN) or cellular fibronectins (cFN) purified from thermolysin digests. A 110-kD fragment (f110), which contains the Arg-Gly-Asp-Ser sequence (RGDS)-dependent cell-binding domain but no heparin-binding domains and whose sequences are shared in common by both the alpha- and beta-subunits of pFN, facilitated attachment of cells that approached the level observed with either intact pFN or the heparan sulfate-binding platelet factor-4 (PF4). This attachment on f110 was resistant to RGDS-containing peptide in the medium. Neurite outgrowth was also maximal on f110, and half of these neurites were also resistant to soluble RGDS peptide. Treatment of cells with glycosaminoglycan lyases failed to alter these responses on f110. Therefore, there is a second "cell-binding" domain in the sequences represented by f110 that is not RGDS- or heparan sulfate-dependent and that facilitates stable attachment and some neurite outgrowth; this domain appears to be conformation-dependent. Comparisons were also made between two larger fragments generated from the two subunits of pFN-f145 from the alpha-subunit and f155 from the beta-subunit--both of which contain the RGDS-dependent cell-binding domain and the COOH-terminal heparin-binding domain but which differ in the former's containing some IIICS sequence at its COOH terminus and the latter's having an additional type III homology unit. Heparin-binding fragments (with no RGDS activity) of f29 and f38, derived from f145 or f155 of pFN, respectively, and having the same differences in sequence, were also compared with f44 + 47 having the "extra domain" characteristic of cFN. Attachment on f145 was slightly sensitive to soluble RGDS peptide; attachment on f155 was much more sensitive. There were also differences in the percentage of cells with neurites on f145 vs. f155 but neurites on either fragment were completely sensitive to RGDS peptide. Mixing of f29, f38, or PF4 with f110 could not reconstitute the activities demonstrated in f145 or f155, demonstrating that covalently linked sequences are critical in modulating these responses. However, mixing of f44 + 47 from cFN with f110 from pFN increased the sensitivity to RGDS peptide.(ABSTRACT TRUNCATED AT 400 WORDS)

Axons↗

PubMed2002

Platelet-binding domains in 2 fibrinogen-binding proteins of Staphylococcus aureus identified by phage display.

The adherence of microorganisms to platelets previously immobilized on the subendocardium in nonbacterial thrombotic endocarditis is considered an important pathogenic step in Staphylococcus aureus endocarditis. To identify and characterize bacterial factors involved in the adherence to platelets, a phage display library of S. aureus was generated by use of the phagemid pG8H6. The library was affinity panned against purified immobilized platelets. After a second panning against platelets, a significant increase in the number of eluted phagemid particles was observed; 27% of 88 randomly isolated clones expressed overlapping deduced amino acid sequences with high similarity to the C-terminal domain of the S. aureus coagulase. In addition, 22% of the clones expressed the N-terminal domain of the fibrinogen-binding protein Efb. The surface-associated fraction of the C-terminal domain of coagulase or the N-terminal domain of Efb may be involved in bacterial adherence to immobilized platelets, and fibrinogen may act as a bridging molecule in that interaction.

Bacterial Adhesion↗

PubMed1997

Profound biotinidase deficiency caused by a point mutation that creates a downstream cryptic 3' splice acceptor site within an exon of the human biotinidase gene.

Biotinidase recycles the vitamin biotin from biocytin upon the degradation of the biotin-dependent carboxylases. We have identified a novel point mutation within the biotinidase gene that encodes the signal peptide in two unrelated individuals with profound biotinidase deficiency. Sequence analysis of genomic DNA from these individuals revealed a G to A transition (G100-->A) located 57 bases downstream of the authentic splice acceptor site in exon B. Although this mutation predicts a G34S substitution, it also generates a 3' splice acceptor site. Sequence of the PCR-amplified cDNA from the homozygous child revealed that all the product was shorter than that of normal individuals and was the result of aberrant splicing. The aberrantly spliced transcript lacked 57 bases, including a second in-frame ATG, that encode most of the putative signal peptide and results in an in-frame deletion of 19 amino acids. The mutation results in failure to secrete the aberrant protein into the blood. This is the first reported example in which a point mutation creates a cryptic 3' splice acceptor site motif that is used preferentially over the upstream authentic splice site. The preferential usage of the downstream splice site is not consistent with the 5'-3' scanning model, but is consistent with the exon definition model of RNA splicing.

Amidohydrolases↗

PubMed1998

A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences.

A simple and efficient ligation-mediated PCR (LMPCR) is described for amplifying DNA adjacent to known sequences. The method uses one primer specific for the known sequence and a second specific for a synthetic linker ligated to restricted genomic DNA. Perkin-Elmer AmpliTaq Gold polymerase is used to minimize non-specific primer annealing and amplification. This LMPCR method was successfully applied to isolate DNA sequences flanking mobile elements present in mycobacterial mutants generated by transposon mutagenesis.

Cloning, Molecular↗

PubMed1989

Identification of a heat-shock pseudogene from Caenorhabditis elegans.

While characterizing the hsp70 gene family from Caenorhabditis elegans we encountered an unusual member of this family. Sequence data reveal that the hsp-2ps gene is a pseudogene of the constitutively expressed, heat-inducible hsp-1 gene. Two stop codons generated near the 5' end of the sequence as well as several frameshift mutations and a large internal deletion confirm the identification of hsp-2ps as a pseudogene. The nucleotide substitution rate of the third codon position was twice that of the first and second codon positions, suggesting that the hsp-2ps gene was nonfunctional since the time of the duplication event. The hsp-2ps gene duplicates a region of the hsp-1 gene that lies exclusively within the transcribed region and retains the introns. We feel that the hsp-2ps gene was produced by a transpositional duplication event, which occurred approximately 8.5 million years ago.

Amino Acid Sequence↗

PubMed2005

[Parathyroid hormone values obtained with immunometric assays depend on the amino-terminal antibody specificity].

Introduction of 2nd generation immunometric assays for the measurement of serum parathyroid hormone (PTH), turned them more available, simple and rapid. These methods, based on double identification of the PTH molecule, supposedly measure the intact, bioactive molecule, with the sequence 1-84. Recent works showed that they also measure forms with amino-terminal deletions, like the 7-84 form, which are not able to activate the traditional PTH receptor (PTH1R). Thus, an important practical aspect is the definition of the PTH forms measured by the immunometric assays, a fact that depends on the specificity of the antibodies employed. In this report we compare the results obtained with an in-house immunofluorometric assay that presents a cross-reactivity of 50% with the 7-84 PTH sequence, and two commercial 2nd generation assays, that react 100%. In a first study, 135 samples were measured using our assay and an electrochemiluminescent assay, resulting in a correlation coefficient of 0.961 (P<0.0001) and medians of 35.0 and 51.0 ng/L (P<0.0001). In a second study, 252 samples were analyzed using our assay and an immunochemiluminometric assay, resulting in a correlation of 0.883 (P<0.0001) and medians of 36.0 and 45.5 ng/L (P<0.0001). In both studies results obtained with the in-house assay were significantly lower, as expected by the specificity of the anti-amino-terminal antibody employed. Our data support the need of a precise description of the specificity of the amino-terminal antibodies employed in 2nd generation PTH assays in order to better compare results and define normal ranges.

Antibody Specificity↗

PubMed1993

Global comparison of radiation and chemotherapy dose-response curves with a test for interaction.

In the analysis of the effects of radiation or drugs on clonogenic survival data of mammalian cells, it is often advantageous to compare entire dose-response curves generated under different experimental conditions rather than to conduct single-dose comparisons. We propose a two-stage method for the global comparison of such curves. The first stage consists of individual fits of a flexible model to the dose-response sequences. The second stage treats the fitted coefficients as data and analyzes them jointly using a multivariate analysis of variance. For dose-response models of the type commonly used in the analysis of clonogenic survival data, this method allows the definition of a statistical test for interaction among treatments. That is, a test that the combination of agents produces a dose-response curve which is not what would be expected if the agents were acting multiplicatively (additively in the log cell survival scale). Analyses of cell survival curves by this method for experiments on iododeoxyuridine-mediated radiosensitization and on chemosensitization of bleomycin cytotoxicity in a human bladder cancer cell line (647V) are presented.

Analysis of Variance↗

PubMed1986

Magnetic resonance imaging of the genitourinary tract.

MRI is in its infancy as a clinical imaging tool. It is undergoing intensive investigation in various areas of the body. Evaluation of the brain and spine is superb, and in some areas of the brain, like the posterior fossa, it is thought to be superior to CT. Evolving indications for body scanning include staging of pelvic malignancies, evaluation of liver malignancy, evaluation and staging of musculoskeletal problems, and, to a lesser degree, staging of renal malignancies and evaluation of vascular disease. The main problem in body imaging stems from image degradation because of respiratory motion that is transmitted to upper abdominal organs. Respiratory gating of image acquisition or utilization of short heavily T1-weighted pulse sequences will likely overcome this problem in due time. Minimizing motion artifact will make MR images comparable to, if not better than, CT images with regard to transverse anatomic display, and MR images have the added advantage of multiplanar scanning, which can be done directly, without need of additional computer reconstruction time and without having to move the patient. The second major problem in MRI is the lack of understanding of equipment potential. Unlike conventional radiography and CT, in which the behavior of the X-ray beam is understood with regard to image formation, in MRI new parameters are used to generate images. As stated earlier, MR signal intensity is due to hydrogen concentration, T1 and T2 relaxation times of the tissue, and flow of protons through the imaged volume. How these factors are weighted depends on pulse sequence selection, and thus image contrast and information content of the scans change. On the surface, these images display anatomic information as do other imaging modalities, but manipulation of pulse sequences may ultimately lead to the ability to demonstrate physiologic and chemical parameters previously unavailable in imaging. Current research is geared to help extract this data by testing new pulse sequences, using different types of receiver RF coils, and using MR-specific contrast materials. Minor MRI problems such as long scan times are being dealt with to decrease time to an acceptable length. The nonvisualization of soft tissue calcifications will probably remain a problem that may have to be weighed against other known advantages. At this time further research and clinical experience are the key to what is needed in MRI, to gain further knowledge with regard to imaging physiologic phenomena, such as flow and spectroscopy, and possibly to monitor the chemical basis of disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcinosis↗

PubMed1993

Anticipation in bipolar affective disorder.

Anticipation refers to the increase in disease severity or decrease in age at onset in succeeding generations. This phenomenon, formerly ascribed to observation biases, correlates with the expansion of trinucleotide repeat sequences (TNRs) in some disorders. If present in bipolar affective disorder (BPAD), anticipation could provide clues to its genetic etiology. We compared age at onset and disease severity between two generations of 34 unilineal families ascertained for a genetic linkage study of BPAD. Life-table analyses showed a significant decrease in survival to first mania or depression from the first to the second generation (P < .001). Intergenerational pairwise comparisons showed both a significantly earlier age at onset (P < .001) and a significantly increased disease severity (P < .001) in the second generation. This difference was significant under each of four data-sampling schemes which excluded probands in the second generation. The second generation experienced onset 8.9-13.5 years earlier and illness 1.8-3.4 times more severe than did the first generation. In additional analyses, drug abuse, deaths of affected individuals prior to interview, decreased fertility, censoring of age at onset, and the cohort effect did not affect our results. We conclude that genetic anticipation occurs in this sample of unilineal BPAD families. These findings may implicate genes with expanding TNRs in the genetic etiology of BPAD.

Adult↗

PubMed1995

IS1-mediated intramolecular rearrangements: formation of excised transposon circles and replicative deletions.

A system is described which permits visualization and analysis of a number of molecular species associated with transposition activity of the bacterial insertion sequence, IS1, in vivo. The technique involves induction of an IS1 transposase gene carried by a plasmid which also includes an IS1-based transposable element. It is, in principle, applicable to the identification of transposition intermediates as well as unstable transposition products and those which are not detectable by genetic means. Thirteen novel molecular species were detected after 4 h of induction. Five major species were characterized, based on their behaviour as a function of time, on their hybridization patterns and on the nucleotide sequences of the transposon-backbone junctions. All result from intramolecular IS1 transposition events. The two reciprocal partner products of IS1-mediated deletions, the intramolecular equivalent of co-integrates generated by intermolecular transposition, have been identified. Both carry a single copy of the transposable element and present complementary distributions of deletion endpoints. These results establish, by direct physical means, that adjacent IS1-mediated deletions are accompanied by duplication of the element. A second type of molecule identified was an excised circular copy of the transposon, raising the possibility that IS1 is capable of following an intermolecular transposition pathway, via excised transposon circles, leading to direct insertion.

Base Sequence↗

PubMed2004

Binding of alpha2-macroglobulin to GRAB (Protein G-related alpha2-macroglobulin-binding protein), an important virulence factor of group A streptococci, is mediated by two charged motifs in the DeltaA region.

GRAB (Protein G-related alpha2M-binding protein) is a surface protein of group A streptococci and exhibits high affinity for alpha2-macroglobulin (alpha2M), a broad-range protease inhibitor. It is the sole alpha2M-binding protein of group A streptococci that has been shown to promote bacterial virulence in a mouse model of skin infection. The binding site for alpha2M was predicted to be in the N-terminal A domain of GRAB. In the present study, the alpha2M-binding domain was first narrowed down to 34 amino acids (amino acids 34-67) using variable truncated N-terminal GRAB fusion proteins. The sequence of the identified domain was used to design overlapping synthetic peptides of different sizes, which were then immobilized on a membrane and assayed for their alpha2M-binding activity. The peptide screening revealed two binding motifs of ten amino acids length, located in the DeltaA (N-terminal part of the A domain) region (amino acids 34-67) with the sequences PRIIPNGGTL (amino acids 41-50) and NAPEKLALRN (amino acids 56-65) respectively. These motifs were used for systematic mutational analysis by generating synthetic peptides containing individual amino acid substitutions at every position of the mapped binding regions. The results indicated a critical role for the arginine residue at position 42 in the first binding domain and at position 64 in the second binding region. Validation of arginine residues as the critical amino acids for alpha2M binding was achieved by site-directed mutagenesis and binding assays. Competitive inhibition assays with GRAB containing amino acid substitutions R42G (Arg42-->Gly), R64G and R42G/R64G indicated differential contribution of the arginine residues at positions 42 and 64 to alpha2M-binding activity and, thus, their involvement in GRAB-induced virulence.

Amino Acid Motifs↗

PubMed1995

Use of PCR-PIRA for screening of a point mutation at codon 12 in K-ras oncogene obtained from paraffin embedded tissue sections.

Among various methods which have been developed for facilitating the screening of point mutations in human genomic DNA, PCR-Primer Introduced Restriction Analysis (PCR-PIRA) is of particular interest due to its practicality and short procedure allowing detection of point mutations by simple restriction enzyme digestion directly after PCR amplification. However, one limitation of PCR-PIRA method is the absence of restriction sites in the region of detection, thus creation of the recognition site in primers has been introduced. Detection of a point mutation at codon 12 in K-ras oncogene by BstNI requires one base change in the primer sequence so that only the normal but not mutant PCR product will be digested by the enzyme. However, false positive results generated from undigested normal DNA sequence are always obtained. This effect is compounded when it is used to analyse mixed cell populations in paraffin embedded section of cancer cells. Assay of a mutant band generated from normal DNA by densitometric quantitation enabled the determination of background values and thereby eliminated false positive results. Samples with higher ratios between mutant and normal bands than the background one after the first PCR-PIRA would be subjected to the second PCR-PIRA in order to confirm the results. Screening of such mutations in cervical carcinomas from paraffin embedded sections using the above criteria should reduce misinterpretation of PCR-PIRA results.

Base Sequence↗

PubMed2003

Decision-making strategies by panic disorder subjects are more sensitive to errors.

BACKGROUND: Decision-making is a complex process, which can be assessed experimentally by the two-choice prediction task. Error-rate, i.e. the frequency of incorrect predictions during this task, is an important factor for the response selection during decision-making. This investigation examined whether the frequency of incorrect predictions has an augmented effect on the number of different strategies underlying decision-making in patients with panic disorder. METHODS: Patients with a DSM-IV diagnosis of panic disorder (PD; N=18), unipolar major depressive disorder (MDD; N=18) and normal comparison subjects (C; N=35) were tested with the two-choice prediction task using three error-rate conditions (20, 50, or 80%). The dynamical entropy of the response sequences was used to quantify the number of different strategies generated during the different error-rates. RESULTS: At 20% error-rates, panic disorder subjects when compared to MDD and C subjects generated more strategies and switched more frequently between strategies as measured by the dynamical entropy and the range of local dynamical entropies. Response bias measures during the two-choice prediction task and post-test self-assessment did not differ between panic disorder subjects and MDD or C subjects. LIMITATIONS: First, panic disorder subjects were medicated. Second, the frequency and intensity of panic attacks and the degree of avoidance behaviors, was not assessed. Third, subjects were tested once only. CONCLUSIONS: Panic disorder subjects show uniformly high response sequence unpredictability in the presence of low error-rates, which is consistent with continued search for an optimal response strategy even when the error-rate is low.

Adult↗

PubMed2005

Determination of CYP2D6 gene copy number by pyrosequencing.

BACKGROUND: Identification of CYP2D6 alleles *5 (deletion of the whole CYP2D6 gene) and *2xN (gene duplication) is very important because they are associated with decreased or increased metabolism of many drugs. The most commonly used method for analysis of these alleles is, however, considered to be laborious and unreliable. METHODS: We developed a method to determine the copy number of the CYP2D6*5 and CYP2D6*2xN alleles by use of Pyrosequencing technology. A single set of PCR and sequencing primers was used to coamplify and sequence a region in the CYP2D6 gene and the equivalent region in the CYP2D8P pseudogene, and relative quantification between these fragments was performed. The CYP2D8P-specific Pyrosequencing peak heights were used as references for the CYP2D6-specific peak heights. RESULTS: Analysis of 200 pregenotyped samples showed that this approach reliably resolved 0-4 genome copies of the CYP2D6 gene. In 15 of these samples, the peak pattern from one analyzed position was unexpected but could be solved by conclusive results from a second position. The method was verified on 270 other samples, of which 267 gave results that corresponded to the expected genotype. One of the samples could not be interpreted. The reproducibility of the method was high. CONCLUSIONS: CYP2D6 gene copy determination by Pyrosequencing is a reliable and rapid alternative to other methods. The use of an internal CYP2D8P control as well as generation of a sequence context ensures a robust method and hence facilitates method validation.

Base Sequence↗

PubMed2005

Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways.

At the onset of M phase, the activity of somatic Wee1 (Wee1A), the inhibitory kinase for cyclin-dependent kinase (CDK), is down-regulated primarily through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase SCF(beta-TrCP). The F-box protein beta-TrCP (beta-transducin repeat-containing protein), the substrate recognition component of the ubiquitin ligase, binds to its substrates through a conserved binding motif (phosphodegron) containing two phosphoserines, DpSGXXpS. Although Wee1A lacks this motif, phosphorylation of serines 53 and 123 (S53 and S123) of Wee1A by polo-like kinase 1 (Plk1) and CDK, respectively, are required for binding to beta-TrCP. The sequence surrounding phosphorylated S53 (DpSAFQE) is similar to the conserved beta-TrCP-binding motif; however, the role of S123 phosphorylation (EEGFGSSpSPVK) in beta-TrCP binding was not elucidated. In the present study, we show that phosphorylation of S123 (pS123) by CDK promoted the binding of Wee1A to beta-TrCP through three independent mechanisms. The pS123 not only directly interacted with basic residues in the WD40 repeat domain of beta-TrCP but also primed phosphorylation by two independent protein kinases, Plk1 and CK2 (formerly casein kinase 2), to create two phosphodegrons on Wee1A. In the case of Plk1, S123 phosphorylation created a polo box domain-binding motif (SpSP) on Wee1A to accelerate phosphorylation of S53 by Plk1. CK2 could phosphorylate S121, but only if S123 was phosphorylated first, thereby generating the second beta-TrCP-binding site (EEGFGpS121). Using a specific inhibitor of CK2, we showed that the phosphorylation-dependent degradation of Wee1A is important for the proper onset of mitosis.

Amino Acid Sequence↗