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PubMed1997

Complete RNA1 sequences of two UK isolates of barley mild mosaic virus: a wild-type fungus-transmissible isolate and a non-fungus-transmissible derivative.

The complete RNA1 sequences of two isolates (fungus transmissible and non-fungus transmissible) of barley mild mosaic virus (BaMMV) were obtained. The two isolates' RNA1 sequences had very high sequence identity (99.3%), and of the 15 amino acid differences (out of 2258) between the putative polyproteins, 11 were conservative and unlikely to affect the structure or function of the protein. The remaining amino acid differences were thought unlikely to affect fungus transmission because they occur in the CI- and NIb-coding regions. This strongly suggests that the P73 protein of RNA2 (which has a 364-aa deletion in the non-fungus-transmissible isolate) is involved in fungus transmission of BaMMV.

Amino Acid Sequence↗
PubMed2003

Experimental infection with the Paderborn isolate of classical swine fever virus in 10-week-old pigs: determination of viral replication kinetics by quantitative RT-PCR, virus isolation and antigen ELISA.

We performed experimental infection in 10-week-old pigs with the Paderborn isolate of classical swine fever virus (CSFV). Despite being epidemiologically linked to the major CSFV outbreak in The Netherlands in 1997, the in vivo replication kinetics of this isolate have to our knowledge not been described in detail previously. We found that oronasal infection with 10(4.7) TCID(50) produced mortality in three out of five pigs after 29-31 days, and severe clinical symptoms in one out of five pigs, while one out of five pigs exhibited no clinical symptoms. At this infection dose, pigs had viral RNA (monitored by quantitative reverse transcription (RT)-PCR) in serum as soon as 2 days post-infection, and excretion of infectious virus (monitored by sentinel pigs) appeared to be virtually concomitant with viremia onset. While virus RNA was cleared from the serum of most pigs after 1-2 weeks, some pigs had viral RNA in serum for more than 30 days, and exhibited only mild clinical symptoms. We observed an excellent correlation between clinical symptoms and viral RNA loads in serum, while serum antibody levels were low. Clinically affected pigs had up to 1000-fold higher serum viral RNA loads than did pigs without clinical symptoms. At this level of infection, and this age group, the Paderborn isolate exhibited a strikingly wide range of replication patterns, which might be relevant to the spread of the virus through susceptible pig populations, and the severity of the 1997-1998 outbreak.

Animals↗
PubMed2001

Evaluation of two current generation enzyme immunoassays and an improved isolation-based assay for the rapid detection and isolation of rotavirus from stool.

BACKGROUND: Rapid and accurate rotavirus testing is important in decisions involving patient care and management. Quality assurance testing needs to be periodically performed, especially among widely used assays having a direct impact on patient care. OBJECTIVES: To evaluate the current generation Kallestad Pathfinder Direct antigen Detection system (PTH), and the widely used Rotaclone(R) Rotavirus EIA Diagnostic Kit (RTC), in comparison with an improved cell culture amplification-antigen detection (CCA-Ag) isolation-based assay. STUDY DESIGN: Two hundred stool specimens (specimen stored at > or =-75 degrees C), which had been previously tested by PTH, were tested by RTC and CCA-Ag. Discordant specimens were retested by PTH, blocking assay, polyacrylamide gel electrophoresis (PAGE), and/or electron microscopy (EM). RESULTS: Among 200 stool specimens, 197 were in accord by PTH, RTC and CCA-Ag. The sensitivity, specificity, positive and negative predictive values for RTC, PTH and CCA-Ag were, 100, 99, 99, 100, 100, 99, 99, 100; and 98, 100, 100, 98%, respectively. Among five initially discordant specimens, two required a period of 10 days to affect isolation. A non-cultivatable (CCA-Ag negative) but true positive specimen, was identified as rotavirus group A serotype G2 by RT-PCR. Four true positive but discordant specimens were blocking assay negative using one or both EIA kits. CONCLUSIONS: PTH and RTC are excellent rotavirus detection system. However, PTH is more expensive (ca. $3.50 vs. $2.00 per test), mandates a slightly longer turn-around time (ca. 1 vs. 1.5 h), and necessitates slightly more hands-on manipulative/preparative steps. Blocking assay was not a reliable confirmatory test for the resolution of specimen discordancy. A combination of CCA-Ag, PAGE, EM, and/or perhaps RT-PCR, is recommended as an appropriate test panel for the resolution of discordant results during assay evaluation. The newly modified and simplified 48-h rotavirus isolation-based assay may serve as a base line methodology in laboratory evalaution studies, as a laboratory support methodology during drug/vaccine efficacy trials, or for the testing of sources (e.g., biopsy/autopsy tissues) not approved for assay by commercial rotavirus kits.

Antigens, Viral↗
PubMed1994

Isolation and microinjection of active sperm nuclei into egg cells and central cells of isolated maize embryo sacs.

Artificial fertilisation was attempted in maize by microinjecting sperm nuclei into the egg cell or central cell of isolated embryo sacs. A protocol for isolation of nuclei from pollen grains was developed and a pure fraction of sperm nuclei was obtained after centrifugation on a Percoll gradient. The in vitro transcriptional activity of the nuclei was tested by incorporation of radioactive UTP into RNA. The level of labelled nucleotide incorporation increased and reached a maximum after between 30 and 40 min in the incubation medium. The embryo sacs were enzymatically isolated and their viability determined by observation of cytoplasmic streaming in the female cells. The embryo sacs were immobilised by embedding in low-melting-point agarose and a single male nucleus was injected with a bevelled microcapillary. The presence of the injected nucleus in the egg or central cell was demonstrated using a cytological approach. This paper presents an alternative method for studying the intimate processes of fertilisation in plants.

Fertilization↗
PubMed2004

Isolation and identification of hexaketides from a pigmented Monosporascus cannonballus isolate.

Monosporascus cannonballus causes severe production losses to muskmelon and watermelon in the United States and other countries. Wild types of the fungus produce no pigments when grown on potato dextrose agar (PDA). After long-term storage on soil/oat hull mix, however, some isolates of the fungus produce yellow to brown pigments and no perithecia when grown on PDA. Five colored metabolites from pigmented cultures of M. cannonballus isolate TX923038 have now been identified. Two of these, monosporascone and dehydroxyarthrinone, have been isolated from other fungi, and three, demethylcerdarin, monosporascol A and azamonosporascone, have not previously been reported. The (1)H NMR and (13)C NMR of all five compounds are reported.

Magnetic Resonance Spectroscopy↗
PubMed2003

Atypical myxomatosis--virus isolation, experimental infection of rabbits and restriction endonuclease analysis of the isolate.

Atypical form of myxomatosis, which caused non-lethal and clinically mild disease in domestic rabbits 1 month after immunization with a commercially available vaccine MXT, is described. The isolated myxoma virus designated as Litovel 2 (Li-2) did not induce systemic disease following subcutaneous and intradermal applications in susceptible experimental rabbits but led to the immune response demonstrated by ELISA. No severe disease was induced in those Li-2 inoculated rabbits by challenge with the virulent strains Lausanne (Lu) or Sanar (SA), while the control animals showed nodular form of myxomatosis with lethal course of the illness. Restriction fragment length polymorphism (RFLP) of genomic DNA with KpnI and BamHI endonucleases was used for genetic characterization of the Li-2 isolate, the vaccine strain MXT and both virulent strains Lu and SA, respectively. In general, RFLP analysis has shown to be informative for inferring genetic relatedness between myxoma viruses. Based on restriction endonuclease DNA fragment size distribution, it was evident that the pathogenic strain SA is genetically related to the reference strain Lu and the isolate Li-2 is more related, but not identical, to the vaccination strain MXT.

Animals↗
PubMed2001

The positive inotropic and chronotropic effects of evodiamine and rutaecarpine, indoloquinazoline alkaloids isolated from the fruits of Evodia rutaecarpa, on the guinea-pig isolated right atria: possible involvement of vanilloid receptors.

Cardiotonic effects of evodiamine and rutaecarpine, constituents of the fruits of Evodia rutaecarpa Bentham Rutaceae, were evaluated on guinea pig isolated atria. Comparison with capsaicin, a vanilloid receptor agonist, revealed similar positive inotropic and chronotropic activity, as judged from antagonistic effects of the competitive vanilloid receptor (capsaicin receptor) antagonist capsazepine, the non-competitive vanilloid receptor antagonist ruthenium red, the calcitonin gene related peptide antagonist CGRP(8-37), the P2X purinoceptor antagonist PPADS, and various desensitization studies. Evodiamine and rutaecarpine produced transient positive inotropic and chronotropic effects on the guinea-pig isolated atria, followed by a desensitizing effect to additional administration. Dose-response relationships for evodiamine, rutaecarpine and capsaicin were obtained. All the compounds evoked positive inotropic and chronotropic effects in a concentration-dependent manner. Maximal contractions for evodiamine, rutaecarpine and capsaicin were observed at concentrations of 1 microM, 3 microM and 0.3 microM, respectively. The cardiotonic responses evoked by both evodiamine and rutaecarpine were shifted to the right by capsazepine, an established antagonist of vanilloid receptor (capsaicin-receptor). The effects of both evodiamine (1 microM) and rutaecarpine (3 microM) were abolished by pretreatment with a desensitizing dosage of capsaicin (1 microM), developing cross-tachyphylaxis between these compounds. The effects of evodiamine (1 microM), rutaecarpine (3 microM) and capsaicin (0.3 microM) were also significantly reduced by pretreatment with ruthenium red (10 microM) and CGRP (8-37) (10 microM). The effects of evodiamine, rutaecarpine and capsaicin were not affected by pretreatment with PPADS (100 microM), a highly selective P2X purinoceptor antagonist, and the possibility of the involvement of the P2X purinoceptor was excluded. These results suggest that the positive inotropic and chronotropic effects on the guinea-pig isolated right atria induced by both evodiamine and rutaecarpine could be attributed to their interaction with vanilloid receptors and the resultant release of CGRP, a cardiotonic neurotransmitter, from capsaicin-sensitive nerves as with capsaicin.

Alkaloids↗
PubMed2003

Pharmacological effects of (+)-nantenine, an alkaloid isolated from Platycapnos spicata, in several rat isolated tissues.

In this work, the potential activity of (+)-nantenine (a natural aporphin alkaloid) in several rat isolated tissues was studied. In rat isolated intact aorta, (+)-nantenine (0.05 - 0.5 microM) competitively antagonized with almost equal effectiveness the contractions produced by phenylephrine (PE) and 5-hydroxytryptamine (5-HT) in normal Krebs solution. However, at higher concentrations (2 microM), the alkaloid also reduced the maximal effect induced by these two agonists. In depolarizing Ca2+-free high KCl 50 mM solution, (+)-nantenine (1.5 - 6 microM) inhibited, in a non-competitive way, the increase in tension evoked by Ca2+ with depression of the maximum response. On the other hand, (+)-nantenine (3 - 30 microM) did not affect the contractile effect caused by okadaic acid (OA, 1 microM) while, however, this alkaloid totally relaxed, in a concentration-dependent fashion, the contractions produced by phorbol 12-myristate 13-acetate (PMA, 1 microM) in endothelium-containing rat aortic rings. (+)-Nantenine (1 - 30 microM) reversed and competitively antagonized the inhibitory action induced by B-HT 920 in electrically-stimulated rat vas deferens. In isolated rat atria, (+)-nantenine (3 - 10 microM) diminished the contraction frequency. (+)-Nantenine (3 microM) significantly reduced the depolarization (voltage)-activated transient (T-type) and sustained (long-lasting, L-type) barium inward currents [ IBa(T) and IBa(L) ] recorded in whole cell-clamped rat aortic myocytes. These results indicate that the pharmacological effects of (+)-nantenine observed at concentrations lower than 1 microM can be attributed to alpha 1 -adrenergic and 5-HT 2A receptor blocking properties whereas at higher concentrations (> 1 microM) the pharmacological activity of this natural compound may be also due to a decrease of Ca2+ influx through transmembrane calcium channels (calcium antagonist activity), to an inhibition of PKC actions and/or to an alpha 2 -adrenoceptor blockade.

Alkaloids↗
PubMed2005

The absorption and excitation spectroscopy of matrix-isolated atomic manganese: sites of isolation in the solid rare gases.

This study collects information from absorption and luminescence excitation spectra recorded for Mn atoms isolated in the solid rare gases Ar, Kr, and Xe and presents an analysis of the site occupancy, based on the polarizabilities of the rare gases and the observed spectral shifts. Two thermally stable sites of isolation exist for atomic Mn in solid Ar and Kr, while a single thermally stable site is present in Mn/Xe. Site occupancy assignments are based on the application of a polarizability model to the z (6)P(5/2)<--a (6)S(5/2); z (8)P(5/2)<--a (6)S(5/2), and y (6)P(5/2)<--a (6)S(5/2) electronic transitions of atomic Mn. From an analysis of the observed RG matrix-to-gas phase energy shifts for P<--S type transitions, this model allows the association of certain site types occupied by metal atoms in the rare gas solids. The required condition being a linear dependence of the matrix shifts with rare gas polarizability for those metal atoms "trapped" in a particular site type. Application of the polarizability model in conjunction with trends observed in site dominance, established a connection between the blue sites in Ar and Kr and the single site in Xe. Use of the known MgRG ground state bond lengths facilitated an identification of the sites of Mn atom isolation assuming the transference of the known MgRG bond lengths to the MnRG systems. Substitutional site occupancy of atomic Mn is assigned to the blue sites in Ar and Kr and the single site in Xe, while tetra-vacancy site occupancy is assigned to the red sites in Ar and Kr. Consistent with these assignments, Mn atoms in solid Ar show a preference for trapping in tetra-vacancy sites whereas in solid Kr, single substitutional sites are preferred and in Xe, this is the only site observed.

Journal Article↗
PubMed1999

Lentogenic field isolates of Newcastle disease virus isolated in Canada and Hungary are identical with the vaccine type used in the region.

Lentogenic field isolates of Newcastle disease virus were examined by restriction enzyme analysis of RT-PCR products generated from the matrix protein gene that discriminates between strains LaSota and B-1, the two most widely used lentogenic vaccine viruses. Isolates were derived from regions where, exclusively or predominantly, only one type of vaccine was employed. Viruses collected in Hungary for two decades were exclusively of LaSota-type while the Canadian collection predominantly included B-1, which corresponded to the vaccine types used in the regions. Isolation of vaccine type lentogenic viruses from unvaccinated flocks supports the occurrence of area spread of these lentogenic viruses.

Journal Article↗
PubMed2005

Possible differences in pathogenicity between cane toad-, frog- and platypus-derived isolates of Mucor amphibiorum, and a platypus-derived isolate of Mucor circinelloides.

Platypuses (Ornithorhynchus anatinus) in the north of the island state of Tasmania, Australia, suffer from a serious disease called ulcerative mycosis, which is responsible for high morbidity and, presumably, mortality rates in areas where it occurs. The disease is caused by the dimorphic fungus Mucor amphibiorum, which is also found in Queensland, New South Wales and Victoria. However, it does not cause disease in platypuses in those states. It has been previously reported that a closely related fungus, Mucor circinelloides, may also be capable of causing this disease. This paper describes pathogenicity trials involving cane toads (Bufo marinus) as the experimental model. The toads were infected with either Tasmanian, platypus-derived M. amphibiorum, West Australian, frog-derived M. amphibiorum, Queensland cane-toad-derived M. amphibiorum or Tasmanian platypus-derived M. circinelloides. The Tasmanian isolates of M. amphibiorum were more likely to cause a serious, long-term infection than were Queensland or West Australian isolates, and (+) mating types caused a more serious infection than the (-) mating type. The isolate of M. circinelloides was incapable of infecting the toads, lending further weight to the theory that it represents an environmental contaminant. The results suggest that an endemic strain of M. amphibiorum has mutated and become pathogenic to platypuses. Alternatively, a pathogenic strain of M. amphibiorum may have been introduced into Tasmania, where it is infecting a naïve population.

Animals↗
PubMed2000

Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus novemcinctus) in an area where the fungus was recently isolated from soil.

Natural infection of armadillos (Dasypus novemcinctus) with Paracoccidioides brasiliensis in Northern Brazil was reported in 1986, raising great interest in the understanding of the role of this mammal in the epidemiological cycle of the fungus. Recently, P. brasiliensis was isolated from the soil of Ibiá, State of Minas Gerais, southeastern Brazil. Armadillos captured in this area were evaluated for the presence of P. brasiliensis in the viscera and infection was detected in 4/16 animals (25%). Fungal yeast phase cells were observed in three of the four infected armadillos by direct microscopic examination and by the indirect immunofluorescence test carried out on homogenized tissues. P. brasiliensis was isolated from three armadillos whose homogenized viscera had been injected into Swiss mice. The new strains (Ibiá-T1, Ibiá-T2 and Ibiá-T3) were identified as P. brasiliensis on the basis of macro- and micromorphology, thermodimorphism, production and serologic activity of exoantigens, and by polymerase chain reaction (PCR)-detection of the gp43 gene. The lethality and lesions caused to the mice from which the strains were recovered confirmed the virulence of the isolates. We conclude that P. brasiliensis infects armadillos in locations with different geoclimatic characteristics and vegetation cover. The direct observation of yeast cells in tissues and the multiple visceral involvement, including the lungs, suggests the occurrence of paracoccidioidomycosis disease in these mammals and supports their role as wild hosts in the epidemiological cycle of the fungus.

Animals↗
PubMed1963

STUDIES ON ISOLATED NUCLEI. I. ISOLATION AND CHEMICAL CHARACTERIZATION OF A NUCLEAR FRACTION FROM GUINEA PIG LIVER.

This article describes a method for the isolation of nuclei from guinea pig liver. It involves the homogenization of the tissue in 0.88 M sucrose-1.5 mM CaCl(2) followed by centrifugation in a discontinuous density gradient in which the upper phase is the homogenate and the lower phase is 2.2 M sucrose-0.5 mM CaCl(2). Based on DNA recovery, the isolated fraction contains 25 to 30 per cent of the nuclei of the original homogenate. Electron microscopical observations showed that approximately 88 per cent of the isolated nuclei come from liver cells (the rest from von Kupffer cells and leucocytes) and that approximately 90 per cent of the nuclei appear intact, with well preserved nucleoli, nucleoplasm, nuclear envelope, and pores. Cytoplasmic contamination is minimal and consists primarily of the nuclear envelope and its attached ribosomes. The nuclear fraction consists of approximately 22.3 per cent DNA, approximately 4.7 per cent RNA, and approximately 73 per cent protein, the DNA/RNA ratio being 4.7. Data on RNA extractibility by phosphate and salt and on the base composition of total nuclear RNA are included.

Animals↗
PubMed2001

Serotype distribution of Salmonella isolates from food animals after slaughter differs from that of isolates found in humans.

If raw meat and poultry are the primary point of entry for Salmonella species into human populations, a correlation might be expected between the serotype distribution of Salmonella species isolated from animals at the time of slaughter and that of isolates found in humans. For 1990-1996, sufficient national data were available to permit such a comparison. A mathematical model was developed to predict serotype distributions of Salmonella isolates among humans on the basis of animal data. There was a significant mismatch between the serotype distributions among humans predicted by the model and those actually observed. This mismatch raises questions about the validity of the "standard" assumptions about Salmonella transmission on which the model was based-namely, that raw animal products are the primary source for human salmonellosis, that the risk of transmission to humans is equal for all food product categories, and that all Salmonella serotypes have an equal ability to cause human illness.

Abattoirs↗
PubMed2005

South African GB virus C isolates: interactions between genotypes 1 and 5 isolates and HIV.

GB virus C (GBV-C) is a common, apparently nonpathogenic human flavivirus that has been associated in some studies with prolonged survival in human immunodeficiency virus (HIV)-positive persons. There are 5 distinct GBV-C genotypes localized to specific geographic regions, and genotype 2 has been previously shown to inhibit HIV replication in vitro in lymphocyte cultures. We identified GBV-C virus isolates representing genotypes 1, 2, and 5 in samples from South African blood donors. GBV-C genotype 1 and 5 isolates replicated in lymphocyte culture, inhibited X4 and R5 HIV-1 isolates, and induced RANTES and stromal-derived factor-1 chemokines in vitro. Thus, African GBV-C genotypes can inhibit HIV replication in vitro.

5' Untranslated Regions↗
PubMed2000

Higher western blot immunoreactivity of glycoprotein 120 from R5 HIV type 1 isolates compared with X4 and X4R5 isolates.

The envelope glycoprotein of human immunodeficiency virus 1 (HIV-1) plays important roles in viral life cycle and pathogenesis. Understanding the immune responses the protein elicits during the course of a viral infection in patients is important in designing an effective vaccine candidate against the virus or for better diagnosis of the disease. In this study, we report that gp120 of R5 isolates have higher Western blot (WB) immunoreactivity to antibodies elicited against the protein in virus-infected human patients compared with that of X4 and X4R5 isolates. Analyses of WB immunoreactivity of chimeric gp120s constructed between R5 (AD8) and X4R5 (DH12) HIV-1 isolates indicate that there are complex tertiary interdomain interactions even after a complete denaturation of the protein. Our data suggest that the determinant(s) responsible for the high WB immunoreactivity might be present in all gp120s, but are accessible to antibodies only for R5 gp120s in the WB assay. The V1/V2 and/or V3 regions of X4 and X4R5 gp120s likely interfere with either the formation or surface exposure of the WB immunoreactive determinant. Supplementing HIV-1 WB diagnosis kits with purified R5 gp120 could improve their sensitivity and facilitate earlier diagnosis of virus infection.

Animals↗
PubMed1979

Synergy between carbenicillin and an aminoglycoside (gentamicin or tobramycin) against Pseudomonas aeruginosa isolated from patients with endocarditis and sensitivity of isolates to normal human serum.

Isolates from the blood of 30 patients with endocarditis due to Pseudomonas aeruginosa were tested for synergy between carbenicillin and an aminoglycoside, either gentamicin or tobramycin, by in vitro checkerboard methods in modified (cation-supplemented) Mueller-Hinton broth. Twenty-five of the 30 isolates were affected synergistically. Whether given low (2.5--5 mg/kg) or high (8 mg/kg) doses of aminoglycoside along with 30 g of carbenicillin daily, all of the five patients infected with pseudomonads that were not synergistically affected were refractory to treatment with pseudomonads that were not synergistically affected were refractory to treatment with the carbenicillin-gentamicin combination, whereas the finding of synergy of carbenicillin with gentamicin (or tobramycin) did not assure a medical cure. Tests for synergy between carbenicillin and gentamicin yielded different results in Mueller-Hinton agar than in modified Mueller-Hinton broth. The majority (28) of 30 isolates of endocarditis-producing P. aeruginosa were resistant to the bactericidal effects of 50% pooled normal serum that had been freshly separated. One of the endocarditis-producing strains that was sensitive to 50% serum was resistant to 10% serum. However, sensitivity or resistance to freshly separated, pooled normal human serum did not predict the outcome of antibacterial therapy for pseudomonas endocarditis.

Aminoglycosides↗
PubMed1992

Characterization of Staphylococcus aureus isolates with decreased susceptibility to vancomycin and teicoplanin: isolation and purification of a constitutively produced protein associated with decreased susceptibility.

"Derivative isolates" with 4- to 8-fold and 8- to 16-fold increases in MICs of vancomycin and teicoplanin, respectively, were selected from 2 susceptible clinical isolates of Staphylococcus aureus by serial incubation in low-level vancomycin. A protein of approximately 39 kDa was demonstrable in the cytoplasmic fraction and occasionally in the membrane fraction by SDS-PAGE of both derivatives. This protein was purified by DEAE chromatography, preparative SDS-PAGE, and electroelution. Derivative bacteria were larger on transmission electron microscopy, had thicker cell walls, and had changes in colony morphology on solid media. Further evidence for cell wall reorganization included loss of phage and capsular typing, decreased susceptibility to lysostaphin/lysozyme killing, and changes in condition for detection of optimal coagulase activity. The mechanism of decreased susceptibility to glycopeptide antibiotics among S. aureus derivative isolates is uncertain. The production of the approximately 39-kDa cytoplasmic protein and cell wall reorganization may mediate changed affinity of glycopeptide-peptidoglycan binding or impairment of glycopeptide access to its cell wall target.

Bacterial Proteins↗