Successive Isolations Research Articles

Structure and chain conformation of water-soluble heteropolysaccharides from Ganoderma lucidum

Five water-soluble heteropolysaccharides designated GL-I to GL-V, were isolated from the cultured fruit body of Ganoderma lucidum. Using a range of techniques the samples are shown to be heteropolysaccharides, mainly composed of glucose, galactose, mannose, arabinose with the relatively low weight-average molecular weights of 6.1×104 to 18.4×104. The amount of uronic acid present is low. As successive isolations proceed to give GL-I to GL-V, the content of glucan increased, the content of galactan and mannan decreased, and the branching decreased from high to near linear. GL-I is most branched of the heteropolysaccharides with a 27.0% degree of branching, whereas GL-V is mainly a linear glucan. Due to their branched chains samples GL-I to GL-III are in a compact coil conformation in 0.9% NaCl at 25°C, whereas GL-V has a slightly flexible chain. Atomic force microscopy images confirmed the spherical shape of samples GL-I to GL-III in water.

The isolation of Corynebacterium coyleae from clinical samples: clinical and microbiological data

Fourteen Corynebacterium coyleae isolates were recovered from 12 in-patients during a 5-years period. In six patients, the isolates were considered as clinically significant, three definite (sepsis), two probable (sepsis and soft tissue infection), and one possible (post-transfusional bacteremia). In the remaining 6 patients (all neonatal bacteremias), there was not enough data for considering the isolates as clinical significant. API Coryne identified all isolates as C. jeikeium, while Biolog GP2 correctly identified 7 out of the 14 isolates. Definitive identification was achieved in all isolates by the sequencing of a fragment of 724 to 1423 pb of 16S rDNA. Successive isolations from two patients presented identical random amplified polymorphic DNA (RAPD) profiles. All of the isolates were in-vitro-sensitive to beta-lactams, gentamicin, rifampin, tetracycline, vancomycin, linezolid, and resistant to clindamycin. Resistance to erythromycin occurred in 83.3% of isolates, all of them presenting phenotype cMLS and harboring the gene ermX.

Basella rugose mosaic virus, a new potyvirus infecting Basella rubra

Plant PathologyVolume 55, Issue 6 p. 819-819 Free Access Basella rugose mosaic virus, a new potyvirus infecting Basella rubra C.-H. Huang, C.-H. Huang Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan 10617, Republic of ChinaSearch for more papers by this authorY.-C. Chang, Corresponding Author Y.-C. Chang *E-mail: ycchang@ntu.edu.tw.Search for more papers by this author C.-H. Huang, C.-H. Huang Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan 10617, Republic of ChinaSearch for more papers by this authorY.-C. Chang, Corresponding Author Y.-C. Chang *E-mail: ycchang@ntu.edu.tw.Search for more papers by this author First published: 09 November 2006 https://doi.org/10.1111/j.1365-3059.2006.01477.xCitations: 7AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Basella rubra and B. alba (Malabar spinach) are plants of the family Basellaceae, native to tropical Asia. They were introduced into Taiwan during the 1660s and are grown as vegetables and herbs. In 2001, mosaic and rugose symptoms were observed on leaves of B. rubra in the Taipei area. Extracts from these plants caused chlorotic lesions on inoculated leaves of Chenopodium amaranticolor and C. quinoa; the latter became systemically infected. A virus isolate, BR, was obtained after three successive single lesion isolations from C. amaranticolor. Following mechanical inoculation, seedlings of B. rubra and B. alba produced symptoms similar to field diseased plants and indicator plants Nicotiana benthamiana and N. clevelandii displayed mosaic symptoms. Transmission electron microscopy of negatively-stained sap extracts showed the presence of flexuous filaments c. 680 nm in length. Using indirect ELISA, plants of B. rubra and C. quinoa with symptoms tested positive for potyvirus using an anti-potyvirus group monoclonal antibody (Agdia Inc.). The only potyvirus previously reported to infect a member of the Basellaceae (Ullucus tuberosus) is Ullucus mosaic virus (Brunt et al., 1982). However this virus differs from isolate BR, as it causes local lesions in C. quinoa. The 3′-terminal region of the BR isolate was amplified by RT-PCR from total RNA of diseased plants, using a potyvirus-specific degenerate primer (PNIbF1) and an oligo-dT primer (Huang & Chang, 2005). A 1·8 kb fragment, including partial NIb, the CP and 3′ UTR, was obtained and cloned. Two independent clones were sequenced and a BLAST search was performed against the NCBI database. Sequence analysis of the putative capsid protein gene revealed that the highest amino acid and nucleotide sequence identities were 70% and 68% respectively, between the BR isolate (DQ394891) and all other searched potyvirus sequences. According to the species demarcation criteria for the Potyviridae (Adams et al., 2005) the BR isolate is a new potyvirus and is herein designated as Basella rugose mosaic virus (BaRMV). References Adams MJ, Antoniw JF, Fauquet CM, 2005. Molecular criteria for genus and species discrimination within the family Potyviridae. Archives of Virology 150, 459– 79CrossrefCASPubMedWeb of Science®Google Scholar Brunt AA, Phillips S, Jones RAC, Kenten RH, 1982. Viruses detected in Ullucus tuberosus (Basellaceae) from Peru and Bolivia. Annals of Applied Biology 101, 65– 71. Wiley Online LibraryWeb of Science®Google Scholar Huang CH, Chang YC, 2005. Identification and molecular characterization of Zantedeschia mild mosaic virus, a new calla lily-infecting potyvirus. Archives of Virology 150, 1221– 30. CrossrefCASPubMedWeb of Science®Google Scholar Citing Literature Volume55, Issue6December 2006Pages 819-819 ReferencesRelatedInformation

A cohort study of Yersinia infection in goats

To determine the temporal pattern of Yersinia infections in three goat flocks and examine the influence of management and seasonal factors on the incidence of those infections over a 1-year period. A longitudinal study involving monthly culture of faeces for Yersinia spp. from age groups of randomly selected goats on three farms in the Manawatu region of New Zealand. The incidence of excretion of potentially pathogenic Yersinia (Yersinia pseudotuberculosis and Y enterocolitica biotypes 2, 3 and 5) peaked in winter and fell in summer. In contrast, environmental Yersinia (Y enterocolitica biotype 1A, Y frederiksenii, Y intermedia and Y rohdei) showed no clear pattern of seasonal variation. Pathogenic Yersinia were more prevalent in young animals than in adults, while environmental Yersinia were more prevalent in adults. The same type was isolated from the same animal in two or more successive months in about 20 to 25% of cases, and in the remaining cases there was a gap of at least one month between successive isolations, with many animals yielding a particular type on only a single occasion. A notable difference was that with the potentially pathogenic types, no animal had more than one period of time when it was found to be excreting a particular type, suggesting that immunity develops following exposure. In contrast, it was common for environmental types to be isolated from the same animal throughout the study period. Two goats were suspected to have developed clinical yersiniosis but all remaining infected animals showed no clinical signs of infection. Asymptomatic Yersinia carriage was common in goats in New Zealand, with a clear seasonal and age group pattern of infection with potentially pathogenic types. There was evidence that immunity developed to potentially pathogenic types. This is the first time that Y rohdei has been isolated from goats.

First report of Hibiscus chlorotic ringspot virus in Taiwan

Hibiscus (Hibiscus rosa-sinesis) is a malvaceous ornamental plant, originating from China, which is commonly grown as a hedge in Taiwan. Virus-like symptoms of chlorotic ringspots surrounded by a yellowish halo are frequently observed on the leaves of hibiscus plants throughout the island. Extracts from these leaves produced chlorotic local lesions when inoculated onto the leaves of Chenopodium quinoa, but did not produce any symptoms on Nicotiana benthamiana or N. clevelandii. After three successive single lesion isolations in C. quinoa, an isolate of a virus was obtained. Transmission electron microscopy (TEM) of negatively stained, partially purified virions revealed the presence of numerous isometric virus particles measuring approximately 28 nm in diameter. According to the literature, two viruses with this particle morphology –Hibiscus chlorotic ringspot virus (HCRSV) and Hibiscus latent ringspot virus (HLRSV) – can induce ringspot symptoms on hibiscus. Using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), these partially purified virions, as well as plants with symptoms, tested positive for HCRSV using a polyclonal antibody (Agdia Inc., Elkhart, IN, USA). In order to further confirm the result of serological test, two specific primers, HCRSV-F1 and HCRSV-R1 were designed according to a published sequence for HCRSV (Huang et al., 2000). Using these two primers, a cDNA fragment of the expected size (3·9 kb) was specifically amplified by reverse transcriptase (RT)-PCR from total RNA extracted from inoculated C. quinoa. Based on the morphology of virions and the results of the ELISA and RT-PCR tests, this virus has been identified as an isolate of HCRSV, a member of the genus Carmovirus. This is the first report of HCRSV and of a carmovirus being identified in Taiwan.

Virological and pathological findings in sheep fetuses following experimental infection of pregnant ewes with cytopathogenic-bovine-virus diarrhoea virus.

Eighteen pregnant Merino ewes were inoculated intravenously between days 65 and 68 of gestation with the unpurified cytopathogenic (cp) bovine virus diarrhoea virus (BVDV) strain Indiana (experiment I). In experiment II, three ewes were inoculated with the same virus after two successive plaque isolations in order to compare its pathogenicity for the fetus with special regard to lesions in the fetal brain. In experiment I, fetal blood and tissue samples, allantoic fluids and placentomes were collected sequentially between 10 and 80 days post-inoculation (p.i.). BVDV was recovered from 6 of 19 fetuses examined during the first 3 weeks after inoculation. From fetuses sampled between 30 and 50 days p.i. virus was isolated from three cases only, and from 60 days p.i. onwards virus was no longer recovered. BVDV was longer detected in the allantoic fluid than in fetal tissues and continued to be present until 80 days post-inoculation. From tissue samples of two fetuses of experiment I, only non-cytopathogenic BVDV was isolated, whilst samples from seven fetuses contained the cp BVDV biotype as revealed by an immunoplaque assay. The cp biotype was also isolated from placentomes. In experiment II, virus was not isolated from any of the tissue samples of two living fetuses collected at 67 days post-inoculation. In both experiments, cp BVDV was recovered from allantoic fluid samples. In contrast to the developing fetal brain, other tissues or organs seemed to be less vulnerable to the cp BVDV strain Indiana. The partial purification of this virus strain did not affect its pathogenicity for the brains of the developing fetuses.

Further Characterization of the Binding of Fibronectin to Gelatin Reveals the Presence of Different Binding Interactions

The specific interaction between fibronectin and collagen has permitted the isolation of fibronectin from plasma using gelatin-Sepharose affinity matrices. In this study we obtained evidence suggesting the presence of an additional high affinity binding interaction between fibronectin and collagen. The following information supports this idea: (i) Successive isolations of fibronectin using the same regenerated gelatin matrix resulted in progressively increased yields of fibronectin, thus indicating the progressive occupation of high affinity fibronectin-binding sites within collagen. (ii) This tightly bound fibronectin could not be eluted from gelatin-Sepharose matrices with strong denaturing agents, such as 8.0 M urea or 6.0 M guanidium chloride. (iii) The fibronectin-occupied matrices supported fibroblast adhesion and spreading, indicating that the amount of tightly bound fibronectin was quantitatively sufficient for this function, and that the fibronectin that bound with high affinity retained its biologically active conformation. (iv) Two fibronectin-derived fragments, CB52 kDa and T55 kDa, selectively bound to fresh gelatin-Sepharose but not to gelatin-Sepharose previously employed to purify fibronectin, suggesting that these fragments recognize only the high affinity binding sites in gelatin. N-terminal amino acid sequence analysis of T55 kDa showed that it contained the previously described collagen-binding domain and included the repeat III-1 of fibronectin. These results support the existence of multiple fibronectin-binding sites in collagen and implicate a high affinity site(s) with a propensity for tenacious binding.

Functional epithelial cell line cloned from rat parathyroid glands.

Primary cultures of rat parathyroid cells were developed in medium containing 5% calf serum, 1% Nutridoma-SP (a serum-free medium supplement from Boehringer Mannheim), and 0.7 mM calcium. The PT-r strain was purified by successive colony isolations and maintained differentiated characteristics (secretion of bioactive and radioimmunoactive parathyroid hormone into the culture medium, sensitivity to calcium regulation, and modulation by secretin) for 7 months in continuous culture. These cultured cells are epithelioid, display diploid chromosome numbers, and do not show a transformed phenotype. There has been no decrease in the rate of cell division or decline in parathyroid hormone secretion since the cell line was established. This clonal cell line provides an important system for further studies on the biology of the parathyroid cell.

Morphological mutant of Rhizoctonia solani and its cell wall component.

The perfect stage of Rhizoctonia solani Kuhn R-44 (Thanatephorus cucumeris) was confirmed by microscopic observation of basidiospores after three successive single spore isolations. A morphological mutant, ML-19, was induced by UV-light irradiation of a mycelial-fragment suspension from a single basidiospore culture of the fourth generation, and characterized. Different from in the cases of some kinds of morphological mutants of Neurospora, the morphology of this mutant was not responsible for conditional morphologicals, such as choline, inositol, linolenic acid and cAMP or hyphal extension factor. The characterization of the cell wall by fractionation, acid hydrolysis, Smith degradation and methylation analysis revealed that fraction III (glycan fraction) was composed of 1, 3-glucomannan containing small amounts of pentoses. The major difference in the fractions III from the original strain and the mutant was the change in the glucose/mannose ratio from 5/3 to 10/3.

Culture of hormone-dependent functional epithelial cells from rat thyroids.

Primary cultures of rat thyroid cells were made in medium supplemented with 0.1--0.5% calf serum and containing six hormones or growth factors: insulin, thyrotropin, transferrin, hydrocortisone, somatostatin, and glycyl-L-histidyl-L-lysine acetate. The FRTL strain was purified by successive colonial isolations and was found to maintain highly differentiated features (secretion into the culture medium of physiological amounts of thyroglobulin and concentration of iodide by 100-fold). The FRTL strain has been observed for more than 3 years in continuous culture. It has maintained the same biochemical and morphological characteristics that typified the primary cultures of thyroid follicular cells immediately after their enzymatic release from the rat thyroid. Thyroid epithelial cells that were grown under more conventional cell culture conditions failed to retain these specialized characteristics. We show that maintenance in vitro of these specialized functions of rat thyroid follicular cells is dependent on low serum concentrations and supplementation with hormones in the primary cultures. Our observations indicate that this culture strategem may be aplicable to the general problem of maintenance of differentiated characteristics in cultures of other epithelial cells.

The establishment of MDMS-resistant cloned cell lines from in vitro cultured, MDMS-sensitive, Yoshida sarcoma cells

A cloned cell line established from an in vivo maintained Yoshida tumour, known to be sensitive to methylene dimethanesulphonate (MDMS), was subjected to selection in vitro by repeated treatments with MDMS. After six successive clonal isolations the resultant clone showed an increase in resistance to MDMS by a factor of 10 3. The cell lines selected in vitro for resistance to MDMS were not crossresistant to HN 2, suggesting that sensitivity to bifunctional alkylating agents is controlled by at least two mutations. The biphasic nature of the dose-response curves even after successive cloning indicates some instability of factors determining resistance.

Temperature-sensitive mutants of human adenovirus type 12.

The availability of conditional lethal viral mutants would be extremely useful in examining the process of reproduction and transformation of the human adeno-virus type 12. This communication de-scribes the isolation of temperature-sensi-tive mutants of adenovirus type 12. The Huie strain of adenovirus type 12 was purified through 3 successive single plaque isolations on human embryonic kidney cell cultures at 37 C. A clari-fied crude virus lysate of this clone, titering1.5†~108 PFU/ml, was subjected to muta-tion in the presence of nitrous acid or hydroxylamine as follows. The virus was added to 10 volumes of prewarmed 1 M acetate buffer (pH 4.0) con-taining 0.75 m nitrous acid [2]. The mix-ture was incubated at 25 C. Every 10 min, aliquots were removed and dialyzed in Dulbecco's phosphate-buffered saline (PBS) and stored at-20 C. The hydroxylamine treatment was performed at 4 C in 0.1 M phosphate buffer (pH 6.0) containing 10-3 M EDTA and 1.6 M hydroxylamine [4]. At indicated intervals, the reaction was stop-ped by diluting an aliquot in 100 volumes of PBS minus Ca + and Mg + containing 10-3 M EDTA and stored at-20 C. Sur-vival was assayed by plaque formation on human embryonic kidney cell cultures at 37 C. As shown in Fig. 1-a and b, treat-ment with either nitrous acid or hydroxyl-

The carrier state of Bacillus subtilis infected with the transducing bacteriophage SP101

Bacteriophage SP10 may infect Bacillus subtilis to form a carrier system. The infected bacteria give rise to infected organisms after six or more successive single-colony isolations. About 60% of the spores derived from such an infected culture yield clones that produce phage, and such spores retain their ability to produce phage even after treatment with antiserum and heating at 80°. Four hours' incubation of carrier spores in nutrient broth yields cultures having more than 10 infective centers per bacterium. Cultivation of infected organisms in medium containing SP10 antiserum leads to loss of phage and loss of immunity to phage infection. DNA with a density characteristic of viral DNA can be detected in infected bacteria. Extracts of infected bacteria contain a phage-induced deoxyribonuclease activity which attacks both phage and bacterial DNA in vitro. The phage-bacterial complex is therefore best described as a carrier state. The DNA of the phage and of B. subtilis are chemically different. Enzymatically prepared complementary virus RNA has no homology with bacterial DNA. The lack of homology of this transducing phage DNA with the DNA of its host and the failure to form a truly lysogenic relationship reinforce previous conclusions that transducing bacterial DNA is incorporated into the SP10 virus particle without any recombination between viral and bacterial DNA.