Comments on "acetaminophen and the cyclooxygenase-3 puzzle: sorting out facts, fictions, and uncertainties".
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Acetaminophen↗
Biomedical subjects
N V Chandrasekharan
Publications and source records attributed to N V Chandrasekharan.
The cyclooxygenases.
Cyclooxygenases (COXs) catalyze the rate-limiting step in the production of prostaglandins, bioactive compounds involved in processes such as fever and sensitivity to pain, and are the target of aspirin-like drugs. COX genes have been cloned from coral, tunicates and vertebrates, and in all the phyla where they are found, there are two genes encoding two COX isoenzymes; it is unclear whether these genes arose from an early single duplication event or from multiple independent duplications in evolution. The intron-exon arrangement of COX genes is completely conserved in vertebrates and mostly conserved in all species. Exon boundaries largely define the four functional domains of the encoded protein: the amino-terminal hydrophobic signal peptide, the dimerization domain, the membrane-binding domain, and the catalytic domain. The catalytic domain of each enzyme contains distinct peroxidase and cyclooxygenase active sites; COXs are classified as members of the myeloperoxidase family. All COXs are homodimers and monotopic membrane proteins (inserted into only one leaflet of the membrane), and they appear to be targeted to the lumenal membrane of the endoplasmic reticulum, where they are N-glycosylated. In mammals, the two COX genes encode a constitutive isoenzyme (COX-1) and an inducible isoenzyme (COX-2); both are of significant pharmacological importance.
Animals↗
COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression.
Two cyclooxygenase isozymes, COX-1 and -2, are known to catalyze the rate-limiting step of prostaglandin synthesis and are the targets of nonsteroidal antiinflammatory drugs. Here we describe a third distinct COX isozyme, COX-3, as well as two smaller COX-1-derived proteins (partial COX-1 or PCOX-1 proteins). COX-3 and one of the PCOX-1 proteins (PCOX-1a) are made from the COX-1 gene but retain intron 1 in their mRNAs. PCOX-1 proteins additionally contain an in-frame deletion of exons 5-8 of the COX-1 mRNA. COX-3 and PCOX mRNAs are expressed in canine cerebral cortex and in lesser amounts in other tissues analyzed. In human, COX-3 mRNA is expressed as an approximately 5.2-kb transcript and is most abundant in cerebral cortex and heart. Intron 1 is conserved in length and in sequence in mammalian COX-1 genes. This intron contains an ORF that introduces an insertion of 30-34 aa, depending on the mammalian species, into the hydrophobic signal peptide that directs COX-1 into the lumen of the endoplasmic reticulum and nuclear envelope. COX-3 and PCOX-1a are expressed efficiently in insect cells as membrane-bound proteins. The signal peptide is not cleaved from either protein and both proteins are glycosylated. COX-3, but not PCOX-1a, possesses glycosylation-dependent cyclooxygenase activity. Comparison of canine COX-3 activity with murine COX-1 and -2 demonstrates that this enzyme is selectively inhibited by analgesic/antipyretic drugs such as acetaminophen, phenacetin, antipyrine, and dipyrone, and is potently inhibited by some nonsteroidal antiinflammatory drugs. Thus, inhibition of COX-3 could represent a primary central mechanism by which these drugs decrease pain and possibly fever.
Acetaminophen↗
Trans-spliced leader RNA, 5S-rRNA genes and novel variant orphan spliced-leader of the lymphatic filarial nematode Wuchereria bancrofti, and a sensitive polymerase chain reaction based detection assay.
A genomic library of Wuchereria bancrofti was examined for the presence of the 22 nucleotide spliced leader (SL) which plays a vital role in the maturation of the 5' end of certain mRNAs through the addition of a small spliced leader (SL) exon and also in the generation of monocistronic mRNA from initial polycistronic transcripts in nematodes. Here, we report the characterization of three SL RNA genes (SLG1, SLG2 and SLG3), an internal copy of a novel variant SL1 sequence (SL1v) with 23 nucleotides within an open reading frame of 75 amino acid residues of an unknown gene and two 5S-rRNA genes (5SR2 and 5SR3) from two genomic clones (TZP/11, TZP/91) of W. bancrofti. Our results revealed that the genes for the spliced leader RNA of W. bancrofti (SL RNA) is reiterated within the 5S-rRNA gene cluster and are in the same orientation. The genes SLG1, SLG2 and SLG3 were identical in nucleotide sequence except for an additional nucleotide at position 43 on SLG2. Sequence analysis of the three genes indicated that the 22-nt sequence is invariably adjacent to the dinucleotide GT, characteristic of a potential spliced donor site. The Sm-binding sequence AATTTTGG was conserved in SLG1, SLG2 and SLG3. Further, both 5' and 3' flanking regions of genes SLG1, SLG2 and SLG3 shared considerable sequence similarity. Two 5S-rRNA genes characterized from the genomic clone TZP 11 were shown to have sequence heterogeneity. Genomic southern showed that the spliced leader sequence is multicopy within the W. bancrofti genome and is also encoded in the region of DNA unlinked to the 5S rRNA gene cluster. Primers designed to amplify intergenic regions between 5S-rRNA and SL RNA genes in a PCR assay were found to be specific for W. bancrofti and was sensitive enough to detect 1 pg of W. bancrofti DNA or 1/8th of a microfilariae in infected blood samples. The high specificity and sensitivity of the optimised PCR assay makes it an ideal diagnostic tool for the identification of W. bancrofti in both the host and the vector.
Amino Acid Sequence↗
Molecular characterisation of the actin gene of the filarial parasite Wuchereria bancrofti.
Wuchereria bancrofti is the major cause of lymphatic filariasis in humans. Although it is responsible for this immensely morbid and debilitating disease, very little is known of the basic molecular biology of this parasite, and there is a vast lack of knowledge on its gene organisation. In this study, the actin gene of W. bancrofti has been characterised by sequencing a clone isolated from a genomic DNA library of this parasite. The 5' flanking region had a potential TATA box and a putative mRNA initiation site. The gene had five exons encoding 376 amino acids, and four introns ranging in size from 109 to 190bp. The 3' flanking region had a potential polyadenylation signal with the sequence ATTAAA which is a common natural variant of the conventional sequence AATAAA. The gene was AT-rich, with a GC content of 37.2%. Southern blot analysis of W. bancrofti genomic DNA indicated that the gene is possibly found as a single copy. The actin amino acid sequence of W. bancrofti showed a high degree of homology to the actin of many organisms of different taxonomic groups, but the highest homology was observed with the free-living nematode Plectus acuminatus. This suggests that P. acuminatus may bear a close evolutionary relationship to W. bancrofti.
Actins↗
Effects of the restriction of food and water intake on the distribution and retention of radioiodine in mice.
The effects of the restriction of food and water intakes on gastrointestinal absorption, distribution to organs and excretion of 131I were investigated in C3H/He mice. The animals were divided into four groups and administered orally 37 kBq carrier-free Na 131I in 0.25 ml normal saline. One group of animals was given food and water ad libitum throughout the experimental period. Food and water to the remaining groups were restricted before and/or after the administration of 131I. The animals in each group were sacrificed 4 h and 24 h after administration, and the activity of 131I in thyroid, blood, liver, kidney, gastrointestinal tract, urine, feces, and carcass was measured. There was a significant increase in the retention of 131I in the thyroid and the concentration of 131I in the blood due to the restriction of food and water after the administration of 131I. In contrast, a significant decrease in the urinary excretion was observed in these animals. In those animals, which fasted before administration only, the retention of 131I in the thyroid and other organs were decreased. Therefore, for an accurate diagnosis and effective therapy with radioiodine as well as effective radiation protection, the intake of food and water should be taken into account.
Animals↗
A polymerase chain reaction based method for the detection of Culex quinquefasciatus (Diptera: culicidae).
Culex quinquefasciatus Say is the major vector of the filarial parasite Wuchereria bancrofti (Cobbold) which causes lymphatic filariasis in humans. A repetitive DNA sequence from the genome of C. quinquefasciatus has been cloned and completely sequenced. The 693 bp cloned fragment had an A+T content of 72%. Dot matrix analysis of the fragment did not reveal any direct or inverted repeats within it. Southern blot analysis using a variety of restriction enzymes appeared to indicate that the cloned fragment was interspersed within the genome with a copy number of approximately 30,000. A search of the GenBank database did not reveal significant homologies to any previously cloned sequences. Although the probe was sensitive enough to detect picogram quantities of DNA, it was not specific for C. quinquefasciatus, as it hybridized with DNA from other mosquito species, Culex pseudovishnui Colless, Culex gelidus Theobald, Culex tritaeniorhynchus Giles, Anopheles vagus Dönitz and Mansonia uniformis (Theobald). However PCR primers derived from the cloned sequence, IpC, were found to be specific and amplified only C. quinquefasciatus DNA. The optimized PCR assay was found to be very sensitive and was capable of detecting DNA from all stages of C. quinquefasciatus thus making it an ideal diagnostic tool.
Animals↗
A sensitive polymerase chain reaction based assay for the detection of Setaria digitata: the causative organism of cerebrospinal nematodiasis in goats, sheep and horses.
A sensitive PCR assay for the detection of Setaria digitata has been developed. Two oligonucleotide primers (17 nt) were designed from a previously cloned and characterized tandemly arranged repetitive sequence of Setaria digitata. Using these primers, it was possible to amplify small quantities (100 fg) of S. digitata genomic DNA. A simple procedure, using proteinase K and non-ionic detergent NP 40, was followed to process the host blood samples and mosquitoes harbouring L3 larvae. The sensitivity of the polymerase chain reaction based assay surpasses the microscopic detection and the previously reported oligonucleotide based chemiluminescent detection of microfilariae in infected host blood samples and L3 larvae in mosquitoes.
Animals↗
Molecular characterisation of a hsp70 gene from the filarial parasite Setaria digitata.
The filarial parasite Setaria digitata is the causative agent of cerebrospinal nematodiasis in its abnormal hosts such as sheep, goats and horses, and therefore is of significant veterinary importance. Since very little is currently known about the biology of this parasite at molecular level, we have cloned and characterised a hsp70 gene, the first gene to be reported from this parasite. The genomic clone isolated contained sequences from two hsp70 genes. One gene, hsp70-2, was completely sequenced and found to contain nine introns ranging in size from 78 to 195 bp. The region upstream of the initiation codon contained a putative TATA box, two CAAT box elements and three heat-shock elements. A putative transcription initiation site was also identified. The 5' untranslated region contained a splice acceptor sequence. The gene was typically AT rich, having a GC content of 44.5%. The deduced aa sequence potentially encoded a cytosolic protein of 645 aa, which had three consecutive repeats of a tetrapeptide motif, GGMP, at the carboxyl end. The gene appeared to be constitutively transcribed and was not significantly enhanced in response to heat shock in adult worms. Another hsp70 gene (hsp70-1) was located further upstream, arranged in direct tandem with hsp70-2. Southern blot analysis revealed the presence of two or three additional hsp70-related genes in the S. digitata genome.
Amino Acid Sequence↗
A simple and rapid non-radioactive oligonucleotide based hybridization assay for the detection of Wuchereria bancrofti.
Five biotin labeled oligonucleotides was designed based on a previously cloned and characterized repetitive DNA sequence specific for Wuchereria bancrofti. The oligonucleotide mix (containing five probes) when used in a hybridization assay, detected as little as 100 pg of purified W. bancrofti, microfilarial DNA, a single infective stage larva and a single microfilaria in 50 microl blood sample. A simple protocol was followed for the hybridization assay. Blood samples lysed with sterile distilled water and digested with proteinase K in the presence of a detergent were directly applied on to nylon membranes for dot blot assays. The DNA extract of mosquitos carrying infective stage larvae was eluted through sephadex G-50 minicolumns prior to blotting. The assay was also able to detect DNA extracted from microfilariae infected samples stored over five days at room temperature (28 degrees C). This simple and rapid oligonucleotide hybridization protocol with the highly sensitive chemiluminescent-based detection has significant potential for the development of a field kit to detect W. bancrofti infection.
Animals↗
Development of a rapid, nonradioactive, oligonucleotide-based assay for the detection of Setaria digitata.
Setaria species are filarial parasites which inhabit the peritoneal cavity of cattle and other ungulates. The parasite is generally considered to be nonpathogenic in its natural hosts, but the transmission of the infective larvae through mosquito vectors to its abnormal hosts (goats, sheep, or horses) can result in a serious and often fatal neuropathological disorder commonly referred to as cerebrospinal nematodiasis. We have previously described the cloning and characterization of a repetitive DNA sequence of Setaria digitata that could be used as a diagnostic probe to detect the parasite in host and vector populations. Here we report the development of a rapid nonradioactive hybridization assay using an oligonucleotide probe based on the above cloned repetitive sequence.
Animals↗
Development of a diagnostic DNA probe to detect Setaria digitata: the causative parasite of cerebrospinal nematodiasis in goats, sheep and horses.
Two repetitive sequences (IpSdM and IpSdS) have been cloned and sequenced from the genome of Setaria digitata. When IpSdM (214 bp) and IpSdS (201 bp) were aligned, a high degree of homology (85%) was observed, indicating that they belong to the same family of repeats. IpSdM represents a complete repeating element while IpSdS consists of two partial repeating elements arranged in tandem. The elements are present in about 10 000 copies comprising 2.8% of the S. digitata genome. As a diagnostic probe IpSdM detects as little as 100 pg DNA of both S. digitata and S. labiato-papillosa. It can also detect a single microfilaria and a L3 larva making it a valuable tool to monitor cattle and mosquito vector populations in the prevention of cerebrospinal nematodiasis.
Animals↗
Polymerase chain reaction-based technique for the detection of Wuchereria bancrofti in human blood samples, hydrocele fluid, and mosquito vectors.
Oligonucleotide primers were designed to amplify a 490-basepair DNA fragment in the 5' end of the pWb 12 repeated DNA sequence in Wuchereria bancrofti for specific amplification of W. bancrofti DNA by the polymerase chain reaction (PCR). A single microfilaria in 100 microliter of blood or added to 1 ml of blood, a single third-stage larva in a pool of 20 uninfected mosquitoes, or 0.4 pg of W. bancrofti genomic DNA added to 100 microliter of human blood or serum can be detected by this PCR method. The parasite DNA in human blood and hydrocele samples and in mosquitoes was isolated free of any PCR inhibitors using simple purification techniques. Detection of PCR products was carried out by agarose gel electrophoresis, followed by staining with ethidium bromide and visualization under ultraviolet illumination. The results indicate that the PCR method is species-specific, rapid, and more sensitive than that of DNA probes and routine microscopy.
Animals↗
Dirofilaria repens: cloning and characterization of a repeated DNA sequence for the diagnosis of dirofilariasis in dogs, Canis familiaris.
A highly repetitive DNA element from the genome of the filarial nematode Dirofilaria repens has been cloned and sequenced. The 176-base pair repeating units are arranged in direct tandem and are clustered in the parasite genome. All repeats appear to belong to a single family although some elements have diverged significantly. The repeats are present in about 15,000 copies and constitute approximately 3.0% of the parasite genome. The cloned repetitive sequence hybridized only to D. repens DNA and was sensitive enough to detect 250 to 500 pg of D. repens DNA, a single microfilariae in infected blood samples, and a single third stage larvae in mosquitoes. The high specificity and sensitivity of the cloned fragment makes it ideal as a diagnostic probe for detecting D. repens in both the host and the vector.
Animals↗
PCR based detection of Mycobacterium tuberculosis: effect of sample preparation.
Tests based on the polymerase chain reaction (PCR) for the detection of the Mycobacterium tuberculosis complex in clinical samples have a lower sensitivity when compared to culture. This has been attributed to the presence of inhibitors to Taq polymerase and/or suboptimal DNA extraction procedures. We tested different methods of processing smear negative culture positive sputum (n = 52) using different detergents, including nonidet P-40 (NP-40), sodium dodecyl sulphate (SDS), tween 20, triton X 100 and N-lauryl sarcosine. The detergents were used in combination with lysozyme and proteinase K enzymes. NP-40 was significantly better than SDS, tween 20 and N lauryl sarcosine (p < 0.05). When NP-40 was used as the detergent, 42 out of 52 specimens gave positive results with the standard amplification protocol which amplifies a 245 bp sequence of the insertion element IS 986. The 10 specimens that were negative were further diluted ten fold and/or eluted in sephadex G-50 columns before standard DNA amplification. A further 8 specimens then became positive. Elution in sephadex G-50 was better than ten fold dilution in processing of samples. The two negative samples had very low colony counts (n < 5). The study demonstrates that the sensitivity of the PCR is dependent on the sample preparation technique and the amount of target sequence available for amplification.
DNA, Bacterial↗
Cloning and characterization of a repetitive DNA sequence specific for Wuchereria bancrofti.
A genomic library constructed in a bacteriophage lambda replacement vector (EMBL3) with Wuchereria bancrofti DNA partially digested with Sau 3A I was screened with 32P-labeled W. bancrofti total DNA, and a strongly reactive recombinant, EMBL3Wb34, was isolated. This clone contained an approximately 16-kb insert that showed some cross-hybridization with Brugia malayi and B. pahangi DNA. However, a 969-bp subclone from EMBL3Wb34, designated pWb12, hybridized only with W. bancrofti DNA and was able to detect as little as 300 pg. Furthermore, pWb12 could detect DNA from a single infective larva or one microfilaria. It has a moderate copy number (450-700) and appears to be interspersed within the parasite genome. The nucleotide sequence contains 66% A+T and 34% G+C and shows no notable internal repeats.
Animals↗