First Report of Seedborne Cherry leaf roll virus in Wild Potato, Solanum acaule, from South America.

A virus, designated JCM-79, was isolated from wild potato (Solanum acaule Bitt.) plants grown from true seed received at USDA-APHIS Potato Quarantine Program from Peru. JCM-79 was mechanically transmissible to Nicotiana clevelandii and N. tabacum cv. Samsun NN. Symptoms in the original S. acaule were general chlorosis and spreading necrotic lesions. Symptoms in N. tabacum and N. clevelandii included necrotic ringspots on inoculated leaves and oak-leaf patterns or necrotic spots, respectively, on upper leaves. Cultivated potatoes (S. tuberosum) infected with JCM-79 by grafting from N. clevelandii were symptomless but virus was detected by back-inoculation to N. clevelandii. Viral nucleoproteins were purified by differential centrifugation and sucrose density gradient fractionation from N. clevelandii and N. tabacum. Transmission electron microscopy of nucleoproteins revealed isometric particles approximately 25 nm in diameter. Two RNA species of approximately 8,000 and 6,500 nucleotides were obtained from nucleoproteins digested with sodium dodecyl sulfate and Proteinase K. The above characteristics suggested JCM-79 was a nepovirus or nepovirus-like in nature. Reverse transcription (RT)-PCR tests for Cherry rasp leaf virus, genus Cheravirus, which was reported from potato (3), were negative. An approximately 1,600-bp cDNA clone was obtained from RNA of JCM-79 by oligo dT primed reverse transcription and second strand cDNA synthesis. Sequence analysis (GenBank No. GU321989) revealed the closest homology (82%) to nucleotides 327 to 1801 of Accession No. S84125 Cherry leaf roll virus (CLRV), genus Nepovirus. Subsequent RT-PCR tests with CLRV-specific primers (4) resulted in amplification of a 417-bp product from nucleic acid extracts of infected N. clevelandii and N. tabacum. The amplified product from N. clevelandii was cloned and three clones were sequenced in both directions. The consensus sequence (GenBank No. GU321988) showed approximately 90% homology to the 3' untranslated region of isolates of CLRV including those from birch, walnut, and sweet cherry (GenBank Nos. S84124, Z34265, and AJ877128, respectively). JCM-79 was also detected in extracts of infected plants by ELISA using CLRV-cherry reagents (Bioreba AG, Reinach, Switzerland). These results indicate JCM-79 represents a new variant of CLRV. To our knowledge, this is the first report of CLRV naturally infecting S. acaule. S. acaule is common in the Andean regions of South America and has been used for crosses with S. tuberosum because of its pathogen resistance (1). The fact that JCM-79 is seed transmitted in S. acaule suggests that this virus could be a threat to potato-breeding programs. Another nepo-like virus with properties similar to JCM-79, designated Potato virus U (PVU), was reported from South America, but PVU was not serologically related to CLRV (2). References: (1) K. Hosaka and D. M. Spooner. Theor. Appl. Genet. 84:851, 1992. (2) R. A. C. Jones et al. Phytopathology 73:195, 1983. (3) J. R. Thompson et al. Arch. Virol. 149:2141, 2004. (4) B. Werner et al. Eur. J. For. Pathol. 27:309, 1997.

Transport and metabolic characterization of Caco-2 cells expressing CYP3A4 and CYP3A4 plus oxidoreductase.

PURPOSE: To further characterize CYP3A4-transfected Caco-2 cells with regard to morphological, transport, and metabolic properties, and to evaluate a different Caco-2 cell strain transfected with both CYP3A4 and oxidoreductase (OR). METHODS: Transfected Caco-2 cells, Caco-2 TC7 cells, and wild-type Caco-2 cells grown onto Millicell were used. We determined the morphological characteristics of transfected cell monolayers using light and transmission electron microscope. We determined the transport and metabolic capabilities of the transfected cells, TC7 cells, and wild-type cells with a variety of drugs, nutrients, and marker compounds. RESULTS: The transfected Caco-2 cells formed a tight monolayer with TEER values and mannitol transport similar to the untransfected parent cell strain (wild type). However, the transfected cells (grown onto Millicell) reached maturity approximately 33% faster than the wild-type cells. Permeabilities of propranolol, nifedipine, testosterone, linopirdine, mannitol, and cephalexin were similar in transfected and wild-type Caco-2 cells. On the other hand, the transfected cells of early passages were much more metabolically active, and metabolized standard CYP3A4 substrates (e.g., testosterone and nifedipine) as much as 100 times faster than untransfected cells. In addition, metabolism of standard substrates was inhibitable by ketoconazole and TAO. Using comparable data, the transfected cells metabolized testosterone the fastest, followed by linopirdine and nifedipine (approximate ratio: 10:6:2). The metabolites of standard substrates were generally preferably excreted to the apical membrane. CONCLUSION: The monolayers of newly transfected cells (CYP3A4 + OR) have a significantly increased level of CYP3A4 activities compared to untransfected cells. These cell monolayers also have desirable morphological and transport characteristics that are similar to untransfected cells.

Human sperm function in co-culture with human, macaque or bovine oviduct epithelial cell monolayers.

Human sperm function was compared in co-culture with monolayers of oviduct epithelial cells (OEC) from three species, human, macaque and bovine. For all species, freeze-thawed and passaged OEC from females in the periovulatory phase were used. OEC cultured on an extracellular matrix (Matrigel) formed a monolayer which supported human sperm attachment to OEC from all three species. Spermatozoa in co-culture with OEC from all three species showed prolonged survival and improved motility characteristics over those cultured in medium alone. This paper describes an efficient, repeatable co-culture system for human spermatozoa which supports sperm attachment to OEC and subsequently improves sperm function over that seen in control medium cultures. Because the improved sperm function in co-culture did not differ significantly between human and bovine OEC for those attributes studied, it is proposed that bovine OEC could be used as an alternative to human OEC in certain human sperm coculture studies. Follicular phase bovine OEC from reproductively normal donors are far more accessible than their human counterparts, thus making this co-culture system more widely available for the study of human spermatozoa-female tract interactions.