Cloning of a full-length cDNA encoding bovine interleukin 4 by the polymerase chain reaction

A human interleukin 4 (hIL-4)-encoding cDNA (hIL4) probe was used to screen a bovine genomic library, and three clones containing sequences with homology to the human and mouse IL4 cDNAs were isolated. Sequence information obtained from one of these genomic clones was used to design an oligodeoxyribonucleotide primer corresponding to the transcription start point region for use in the polymerase chain reaction (PCR). The PCR-RACE protocol, designed for the rapid amplification of cDNA ends, was successfully used to generate a full-length bovine IL4 (bIL4) cDNA clone from polyadenylated RNA isolated from concanavalin A-stimulated bovine lymph node cells. The bIL4 cDNA is 570 bp in length and contains an open reading frame of 405 nucleotides (nt), coding for a 15.1-kDa precursor of 135 amino acids (aa), which should be reduced to 12.6 kDa for unglycosylated bIL4 after cleavage of a putative hydrophobic leader sequence of 24 aa. The aa sequence contains one possible Asn-linked glycosylation site. Bovine IL4 is shorter than mouse (mIL4) and hIL4, because of a 51-nt deletion in the coding region. Comparison of the overall nt and deduced aa sequences shows a greater homology of bIL4 with hIL4 than with mIL4. This homology is not evenly distributed, however, with the nt sequences 5' and 3' of the coding region showing a much greater homology between all three species than the coding sequence.

Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction

Degenerate oligonucleotide primers derived from conserved cysteine protease sequences were used in the reverse transcription polymerase chain reaction to amplify seven different cysteine protease cDNA clones, Fcp1-7, from RNA isolated from adult Fasciola hepatica. Five of the amplified F. hepatica sequences showed homology to the cathepsin L type and two were more related to the cathepsin B type. Southern blot analysis suggests that some members of this protease gene family are present in multiple copies. Northern blot analysis revealed differences in the levels of steady state mRNA expression for some of these proteases. The 5' and the 3' regions of Fcp1 were amplified using the rapid amplification of cDNA ends PCR protocol (RACE-PCR) and an additional clone was obtained by screening a lambda gt10 cDNA library using Fcp1 as a probe. The Fcp1 cDNA fragment was also subcloned in the expression vector pGEX and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli. Antibodies, raised in rabbits against the GST:Fcp1 fusion protein, were used in western blot analysis to examine expression in different life-cycle stages of F. hepatica. In extracts from adult and immature parasites, the immune serum recognised predominantly two proteins of 30 kDa and 38 kDa. In other parasite stages, proteins of different molecular weight were recognised by the anti-GST:Fcp1 antiserum, indicating stage-specific gene expression or processing of Fcp1. In gelatine substrate gel analysis, strong proteolytic activity could be detected at 30 kDa, but not at 38 kDa, suggesting that the 30 kDa protein represents the mature enzyme and the 38 kDa protein the proenzyme.

Identification and characterization of a liver stage-specific promoter region of the malaria parasite Plasmodium.

During the blood meal of a Plasmodium-infected mosquito, 10 to 100 parasites are inoculated into the skin and a proportion of these migrate via the bloodstream to the liver where they infect hepatocytes. The Plasmodium liver stage, despite its clinical silence, represents a highly promising target for antimalarial drug and vaccine approaches. Successfully invaded parasites undergo a massive proliferation in hepatocytes, producing thousands of merozoites that are transported into a blood vessel to infect red blood cells. To successfully develop from the liver stage into infective merozoites, a tight regulation of gene expression is needed. Although this is a very interesting aspect in the biology of Plasmodium, little is known about gene regulation in Plasmodium parasites in general and in the liver stage in particular. We have functionally analyzed a novel promoter region of the rodent parasite Plasmodium berghei that is exclusively active during the liver stage of the parasite. To prove stage-specific activity of the promoter, GFP and luciferase reporter assays have been successfully established, allowing both qualitative and accurate quantitative analysis. To further characterize the promoter region, the transcription start site was mapped by rapid amplification of cDNA ends (5'-RACE). Using promoter truncation experiments and site-directed mutagenesis within potential transcription factor binding sites, we suggest that the minimal promoter contains more than one binding site for the recently identified parasite-specific ApiAP2 transcription factors. The identification of a liver stage-specific promoter in P. berghei confirms that the parasite is able to tightly regulate gene expression during its life cycle. The identified promoter region might now be used to study the biology of the Plasmodium liver stage, which has thus far proven problematic on a molecular level. Stage-specific expression of dominant-negative mutant proteins and overexpression of proteins normally active in other life cycle stages will help to understand the function of the proteins investigated.

The platelet collagen receptor glycoprotein VI is a member of the immunoglobulin superfamily closely related to FcalphaR and the natural killer receptors

We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.

A novel family of transporters mediating the transport of glutathione derivatives in plants

Uptake and compartmentation of reduced glutathione (GSH), oxidized glutathione (GSSG), and glutathione conjugates are important for many functions including sulfur transport, resistance against biotic and abiotic stresses, and developmental processes. Complementation of a yeast (Saccharomyces cerevisiae) mutant (hgt1) deficient in glutathione transport was used to characterize a glutathione transporter cDNA (OsGT1) from rice (Oryza sativa). The 2.58-kb full-length cDNA (AF393848, gi 27497095), which was obtained by screening of a cDNA library and 5'-rapid amplification of cDNA ends-polymerase chain reaction, contains an open reading frame encoding a 766-amino acid protein. Complementation of the hgt1 yeast mutant strain with the OsGT1 cDNA restored growth on a medium containing GSH as the sole sulfur source. The strain expressing OsGT1 mediated H-3]GSH uptake, and this uptake was significantly competed not only by unlabeled GSSG and GS conjugates but also by some amino acids and peptides, suggesting a wide substrate specificity. OsGT1 may be involved in the retrieval of GSSG, GS conjugates, and nitrogen-containing peptides from the cell wall.

Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 10(3) and 5 × 10(4) cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Different disparities of gender and race among the thrombotic thrombocytopenic purpura and hemolytic-uremic syndromes

Thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS) represent multiple disorders with diverse etiologies. We compared the gender and race of 335 patients enrolled in the Oklahoma TTP-HUS Registry across 21 years for their first episode of TTP or HUS to appropriate control groups. The relative frequency of women and white race among patients with TTP-HUS-associated with a bloody diarrhea prodrome and the relative frequency of women with quinine-associated TTP-HUS were significantly greater than their control populations. The relative frequency of women and black race among patients with idiopathic TTP and TTP-associated with severe ADAMTS13 deficiency was significantly greater than their control populations. The relative frequency of black race among patients who had systemic lupus erythematosus (SLE) preceding TTP was significantly greater than among a population of patients with SLE, and the relative frequency of black race among patients with other autoimmune disorders preceding TTP was significantly greater than their control population. No significant gender or race disparities were present among patients with hematopoietic stem cell transplantation-associated thrombotic microangiopathy, TTP associated with pregnancy, or TTP associated with drugs other than quinine. The validity of these observations is supported by the enrollment of all consecutive patients across 21 years from a defined geographic region, without selection or referral bias. These observations of different gender and race disparities among the TTP-HUS syndromes suggest the presence of different risk factors and may serve as starting points for novel investigations of pathogenesis.

Rapid assessment of internodal myelin integrity in central nervous system tissue

Monitoring pathology/regeneration in experimental models of de-/remyelination requires an accurate measure not only of functional changes but also of the amount of myelin. We tested whether X-ray diffraction (XRD), which measures periodicity in unfixed myelin, can assess the structural integrity of myelin in fixed tissue. From laboratories involved in spinal cord injury research and in studying the aging primate brain, we solicited "blind" samples and used an electronic detector to record rapidly the diffraction patterns (30 min each pattern) from them. We assessed myelin integrity by measuring its periodicity and relative amount. Fixation of tissue itself introduced +/-10% variation in periodicity and +/-40% variation in relative amount of myelin. For samples having the most native-like periods, the relative amounts of myelin detected allowed distinctions to be made between normal and demyelinating segments, between motor and sensory tracts within the spinal cord, and between aged and young primate CNS. Different periodicities also allowed distinctions to be made between samples from spinal cord and nerve roots and between well-fixed and poorly fixed samples. Our findings suggest that, in addition to evaluating the effectiveness of different fixatives, XRD could also be used as a robust and rapid technique for quantitating the relative amount of myelin among spinal cords and other CNS tissue samples from experimental models of de- and remyelination.

Rapid induction of remission in large vessel vasculitis by IL-6 blockade. A case series

OBJECTIVE: To evaluate the effect of IL-6 blockade using tocilizumab in inducing remission of arterial large vessel vasculitides (LVV). METHODS: Five consecutive patients with giant-cell arteritis (GCA) and two with Takayasu’s arteritis (TA) were treated by tocilizumab infusions (8 mg/kg). Tocilizumab was given every other week for the first month and once monthly thereafter. Clinical symptoms of disease activity, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level and glucocorticoid (GC) dosage necessary to maintain remission were prospectively assessed. MR angiography was performed to monitor local inflammation. RESULTS: Of the seven patients three were newly diagnosed and four showed GC resistance, i.e. GC could not be lowered to less than 7.5 mg/day. The mean follow-up time was 4.3 months (range 3–7 months). All patients achieved a rapid and complete clinical response and normalisation of the acute phase proteins. Remarkably, prednisone dosage could be reduced within 12 weeks to a mean of 2.5 mg/day (range 0–10 mg/day). No relapse and no drug-related side effects were noted. CONCLUSION: Collectively the data suggest that IL-6 blockade using tocilizumab qualifies as a therapeutic option to induce rapid remission in large vessel vasculitides.

Rapid adaptation of visual search in simulated hemianopia

Patients with homonymous hemianopia have altered visual search patterns, but it is unclear how rapidly this develops and whether it reflects a strategic adaptation to altered perception or plastic changes to tissue damage. To study the temporal dynamics of adaptation alone, we used a gaze-contingent display to simulate left or right hemianopia in 10 healthy individuals as they performed 25 visual search trials. Visual search was slower and less accurate in hemianopic than in full-field viewing. With full-field viewing, there were improvements in search speed, fixation density, and number of fixations over the first 9 trials, then stable performance. With hemianopic viewing, there was a rapid shift of fixation into the blind field over the first 5-7 trials, followed by continuing gradual improvements in completion time, number of fixations, and fixation density over all 25 trials. We conclude that in the first minutes after onset of hemianopia, there is a biphasic pattern of adaptation to altered perception: an early rapid qualitative change that shifts visual search into the blind side, followed by more gradual gains in the efficiency of using this new strategy, a pattern that has parallels in other studies of motor learning.

A pyrosequencing assay for the rapid discrimination of mitochondrial lineages in the Salmo trutta species complex

The European trout (Salmo trutta species complex) is genetically very diverse consisting of five distinct mitochondrial lineages that probably originated in the Pleistocene. Here, we describe a novel pyrosequencing protocol to generate two short sequence reads from the mitochondrial control region, which allow the unambiguous identification of all five lineages. The approach was found to be easily transferable between laboratories and should be a valuable tool for the assessment of genetic diversity in trout. Pyrosequencing-based assays for molecular species identification are expected to be generally useful whenever multiple positions in a short DNA sequence need to be assessed.