Cultivation and partial characterization of spiroplasmas in cell cultures.

Spiroplasmas were propagated in the Drosophila melanogaster cell line Dm-1. Spiroplasma citri and unidentified strains (corn shunt organism, 277F [tick isolate], powder puff, BNR-1, honey bee, and OBMG) grew to 10(8) to 10(9) colony-forming units per ml and could be passaged. Cytopathic effect (CPE) varied with the infecting spiroplasma. The honey bee isolate killed Dm-1 within 2 to 4 days and produced CPE in four mammalian cells tested. At 25 degrees C, suckling mouse cataract agent produced no CPE in Dm-1 cells. Dm-1 cells did not support growth of the spiroplasmal sex ratio organism. Spiroplasmas could be detected in the cell cultures by agar inoculation, dark-field microscopy, scanning electron microscopy, and DNA fluorescent staining. The uridine phosphorylase test showed significant levels of conversion of [14C]uridine to [14C]uracil for all but some plant isolates: S. citri, corn shunt organism, lettuce, cactus, and powder puff strains, the first mycoplasmas to lack the enzyme. Primary isolations of corn shunt organism from infected corn plants were made in Dm-1 and I-XII cultures. The course of corn stunt organism infection of Dm-1 was monitored for three passages. The use of agarose and Dienes staining of the colonies improved growth and colony counting of corn stunt organism. The number of viable infected DM-1 cells decreased from 1.2 x 10(7) at passage 1 to 7.0 x 10(6) at passage 2 and 3 x 10(5) at passage 3.     

Cell cycle traverse in AHH-1 tk +/− human lymphoblastoid cells exposed to the chromosomal mutagen,m-amsa

AHH-l tk +/− cells were exposed to the chemotherapeutic agent, m-amsa, both in complete medium and in medium without serum, subcultured in complete medium, and the effect on the traverse of the cell cycle determined by flow cytometric analysis of bromodeoxyuridine (BrdUrd)-labeled DNA. After exposure to m-amsa (day 0), the percentage of S-phase cells increased significantly (P < 0.0017) with increasing concentration. Cells also accumulated in G2/M as evidenced by the significant (P < 0.0026), concentration-dependent increase in the percentage of cells detected within this phase. Serum deprivation during exposure resulted in significantly (P = 0.024) more cells in S-phase than in cultures exposed to m-amsa in complete medium. After three days in culture, a significant (P = 0.0001) accumulation of cells in G2/M was present; the percentage of cells in G2/M did not differ significantly (P = 0.148) in cultures exposed to m-amsa in complete medium or in serum-free medium. However, a significant (P < 0.001) loss of S-phase cells was found in cultures exposed without serum. At day 7, no significant concentration effects were detected (G0/G1, P = 0.6026; S-phase, P = 0.9773; G2/M, P = 0.8401). These results demonstrate that exposure to m-amsa perturbs the traverse of the cell cycle, initially by inhibiting the completion of S-phase and followed by an accumulation of cells in G2/M. In addition, exposure to m-amsa under conditions of serum deprivation results in an increased percentage of cells in the initial S-phase after exposure, the loss of S-phase cells from the culture after three days, and the appearance of a subdiploid peak, consistent with cells undergoing apoptosis. © 1996 Wiley-Liss, Inc.     

Somatic mutation screening: identification of individuals harboring K-ras mutations with the use of plasma DNA

BACKGROUND: Many cancers are attributed to somatic mutation of DNA. We investigated whether it is feasible to detect cancer-associated somatic mutations in patients with neoplasms by using plasma DNA. METHODS: Plasma samples were prospectively collected from 240 patients undergoing colonoscopy. Colorectal biopsies were performed as clinically indicated in 135 patients, and risk factor information was available from 232 patients. DNA was extracted from plasma and colorectal tissue and was amplified by use of a polymerase chain reaction method that enriches for mutations in codon 12 of the K-ras oncogene. Molecular, histologic, and clinical data were compared by use of two-sided Fisher's exact test. RESULTS: Mutations in the K-ras gene detected in the plasma of 64 (28%) of 232 patients were statistically significantly associated with colorectal cancer risk factors (P =.0002). Of those patients having tissue available for comparison (n = 135), mutations in the K-ras gene were found in the tissues of 35 patients, and 29 (83%) of these 35 showed mutations in plasma samples. In contrast, the plasma assay was negative in 93 of the 100 patients whose tissue K-ras was wild-type. Among patients without biopsies (n = 105), 28 had mutated K-ras in their plasma DNA, despite the absence of remarkable colonoscopy findings; 24 of these 28 patients had risk factors for colorectal cancer. Overall, 25 (39%) of 64 patients showing mutations in plasma DNA had colorectal neoplasms with K-ras mutations compared with five (3%) of 176 patients without K-ras mutations in plasma DNA. CONCLUSION: Plasma DNA assays for the detection of mutations in K-ras codon 12 may provide a feasible method to screen populations for somatic mutations frequently found in neoplasms. The clinical utility of using this test in screening populations requires further study.     

Fumonisin B1 induces apoptosis in cultured human keratinocytes through sphinganine accumulation and ceramide depletion

Fumonisin B1 stimulates apoptosis in a variety of cell types and tissues. We examined the role of sphingolipid changes in fumonisin B1-stimulated apoptosis. Sphinganine accumulated rapidly, sphingosine levels remained unchanged, and ceramides decreased during fumonisin B1 exposure. Increased DNA fragmentation, decreased viability, and apoptotic morphology were observed in cells exposed to fumonisin B1, sphinganine, or N-acetylsphingosine. Co-exposure to N-acetylsphingosine or beta-chloroalanine, which blocks sphinganine accumulation, partially protected cells from fumonisin B1-induced apoptosis. These results illustrate three sphingolipid-dependent mechanisms for inducing apoptosis: accumulation of excess ceramide, accumulation of excess sphinganine, and depletion of ceramide or complex sphingolipids derived from ceramide.     

Molecular evaluation of biopsy and autopsy specimens from patients receiving in vivo retroviral gene therapy

We used the polymerase chain reaction (PCR) to assay for the presence of retroviral vector and replication-competent retrovirus (RCR) in autopsy and biopsy specimens from patients who received inoculations of retroviral vector producer cells (VPCs) into brain tumors or apparently normal tissues surrounding resected tumors. The PCR assays were capable of detecting 1 or more proviral copies of vector or RCR in 500,000 cells. Of 113 patients treated in clinical trials between 1994 and 1997, autopsy specimens were available from 32 patients. Brain tumor biopsies were also available from 24 patients. A total of 346 specimens was analyzed. Vector DNA was detected in 55% of tumor samples and 22% of brain samples obtained from resection margins. In contrast, most of the nonbrain tissues were negative for vector DNA; only low levels (<0.03%) of vector sequence were detected in 6 of 240 (2.5%) nonbrain tissues. Vector DNA was not detected in gonadal tissues from 12 men and 10 women. More importantly, RCR was not detected in any of the 134 biopsy and autopsy tissues tested, including all brain tumor, brain, and gonadal specimens. These results comprise the largest data set on molecular analysis of autopsy specimens from patients receiving retroviral gene therapy and indicate that distribution of retroviral vectors following injection of high doses of VPCs is limited to the site of inoculation.