Localization of integrated adenovirus DNA in the hamster genome

Adenovirus DNA integrated into the genomes of adenovirus-transformed hamster cells or of adenovirus type 12 (Ad12)-induced hamster tumor cells can be located at many different chromosomal sites. This raises the question as to whether distinct isochores of the hamster cell genome might be more accessible to recombination with adenovirus DNA. In Ad12- or Ad2-transformed hamster cell lines, and in Ad12 revertants, the investigated integrated viral DNA sequences were assigned to isochore families by analyzing DNA fractions from preparative CsCl density gradients for their buoyant densities (and, therefore, GC levels) and for the presence of viral DNA. Adenovirus DNA sequences were found in different isochores, which did not generally match the base composition of viral sequences. This is in apparent contrast to what was previously observed with retroviral integration. However, in cell lines carried in culture for many years, the viral DNA sequences might have been transposed to different isochore positions, since the host sequences flanking the viral DNA appear to have been conserved.Received 6 July 2004; received after revision 23 August 2004; accepted 6 October 2004     

Effects of vitamin B12 on plasma melatonin rhythm in humans: increased light sensitivity phase-advances the circadian clock?

Vitamin B12 (methylcobalamin) was administered orally (3 mg/day) to 9 healthy subjects for 4 weeks. Nocturnal melatonin levels after exposure to bright light (ca. 2500 lx) were determined, as well as the levels of plasma melatonin over 24 h. The timing of sleep was also recorded. Vitamin B12 was given blind to the subjects and crossed over with placebo. We found that the 24-h melatonin rhythm was significantly phase-advanced (1.1 h) in the vitamin B12 trial as compared with that in the placebo trial. In addition, the 24-h mean of plasma melatonin level was much lower in the vitamin B12 trial than with the placebo. Furthermore, the nocturnal melatonin levels during bright light exposure were significantly lower in the vitamin B12 trial than with the placebo. On the other hand, vitamin B12 did not affect the timing of sleep. These findings raise the possibility that vitamin B12 phase-advances the human circadian rhythm by increasing the light sensitivity of the circadian clock.     

Effects of vitamin B12 on plasma melatonin rhythm in humans: Increased light sensitivity phase-advances the circadian clock?

Vitamin B12 (methylcobalamine) was administered orally (3 mg/day) to 9 healthy subjects for 4 weeks. Nocturnal melatonin levels after exposure to bright light (ca. 2500 lx) were determined, as well as the levels of plasma melatonin over 24 h. The timing of sleep was also recorded. Vitamin B12 was given blind to the subjects and crossed over with placebo. We found that the 24-h melatlonin rhythm was significantly phase-advanced (1.1. h) in the vitamin B12 trial as compared with that in the placebo trial. In addition, the 24-h mean of plasma melatonin level was much lower in the vitamin B12 traial than with the placebo. Furthermore, the nocturnal melatonin levels during bright light exposure were significantly lower in the vitamin B12 trial than with the placebo. On the other hand, vitamin B12 did not affect the timing of sleep. These findings raise the possibility that vitamin B12 phase-advances the human circadian rhythm by increasing the light sensitivity of the circadian clock.     

Activity of soluble factors produced by Epstein-Barr virus-transformed human B lymphocytes on different cell lines.

Self-stimulatory growth factors, produced by a human Epstein-Barr virus (EBV)-positive lymphoblastoid B cell line, named BA-D10-4, have been tested for the capacity to induce DNA synthesis in various human and animal cell lines, including lymphoid, either EBV-positive or EBV-negative, and non-lymphoid cell lines. It has been found that BA-D10-4 cells produce growth factors which seem to be essential for their sustained proliferation in vitro, and which increase DNA synthesis in different primate lymphoid cells, independently of the presence of the EBV genome and of the lymphocyte lineage.     

Activity of soluble factors produced by Epstein-Barr virus-transformed human B lymphocytes on different cell lines

Summary Self-stimulatory growth factors, produced by a human Epstein-Barr virus (EBV)-positive lymphoblastoid B cell line, named BA-D10-4, have been tested for the capacity to induce DNA synthesis in various human and animal cell lines, including lymphoid, either EBV-positive or EBV-negative, and non-lymphoid cell lines. It has been found that BA-D10-4 cells produce growth factors which seem to be essential for their sustained proliferation in vitro, and which increase DNA synthesis in different primate lymphoid cells, independently of the presence of the EBV genome and of the lymphocyte lineage.     

The structure of amoorastatone and the cytotoxic limonoid 12-hydroxyamoorastatin.

2 new limonoid-type terpenes have been isolated from an aqueous extract of seeds produced by the Eastern Himalayan (India) plant Aphanamixis grandifolia B1. By interpreting principally mass spectral and nuclear magnetic resonance data, the structures of 12-hydroxyamoorastatin (2b) and amoorastatone (3) were elucidated. Unequivocal evidence for the 12-hydroxyamoorastatin structural assignment was obtained by chemical conversion to sendanin (4). Amoorastatin derivative 2b was found to significantly inhibit growth of the murine P388 lymphocytic leukemia cell lines but amoorastatone in the same system was inactive. In a comparative biological study, sendanin (4) and anthothecol (7) were also found significantly to inhibit growth of the P388 cell line, while rohitukin (8) and limonin (9) were found to be inactive.     

Establishment of normal diploid and malignant heteroploid cell lines from non-treated and benzo(a)pyrene treated hamster embryo cell cultures

Summary Two normal diploid control cell lines and a heteroploid malignant transformed cell line from B(a) P treated hamster embryo cell cultures were established. The 14-month-old B(a)P transformed cell line grew 8-times faster than the 20-month-old control cell line. The control cell line showed normal diploid chromosome complement in 93% cells and heteroploidy in 7% cells while B(a)P treated line showed 83% heteroploid cells and only 17% diploid cells. This is the first report on the establishment of diploid hamster cell cultures grown for extended period. Acknowledgments. This work was supported by the French Ministry of Quality of Life, by the Institut National de la Santé et Recherche Médicale, by the S.E.I.T.A. and by Contract No. 12-14-7001-297 of the Agricultural Research Service, U.S. Dept. of Agriculture, Administered by the Athens, Georgia Area, Richard B. Russell Agricultural Research Center, Athens, Georgia 30604, USA. We thank Mr.B. R. Sharma, Mr.E. J. Radin, Dr.R. Hakim and Dr.Ved Brat for their help during this work.     

Demonstration of the presence of M-creatine kinase in mammalian myogenic cell lines

Summary Unequivocal identification of M-CK in cell extracts from fused cells of myogenic cell lines is difficult due to almost identical behaviour of the M-CK and a contaminating enzyme activity in electrophoresis. If CK dimers present in cell extracts were subjected to dissociation and reassociation in the presence of exogenous B-CK subunits, the formation of easily identifiable MB-CK was demonstrated, indicating the presence of M-CK in the myogenic rat cell lines.Acknowledgments. We are grateful to Mrs M. Siegrist and Dr D. Turner for communication of preliminary results and helpful discussions, Mrs E.R. Perriard for skillful technical help. Supported by a grant of the Muscular Dystrophy Association Inc. to H.M.E.     

Vitamins and other metabolites in various sera commonly used for cell culturing

Many cell culture media use different sera to enhance growth. We assayed vitamins and some related metabolites in different sera and identified the concentration of: thiamin, biotin, folates, riboflavin, pantothenates, nicotinates, vitamins B6, B12, A, E, C, and carotenes and some related metabolites: biopterins, free inositol, free and total choline, total carnitines in chicken, horse, rabbit, goat, pig, calf, newborn calf, fetal calf and human sera. Results indicate that vitamin and metabolite content of different sera vary. Such variations could produce fluctuant effects on cell culturings if the metabolite content of the serum is not documented.     

Karyological studies on established mosquito cell lines

Chromosome frequency distribution and cellular DNA estimations in different established mosquito cell lines were studied. These cell lines exhibited a wide range of cell types with a diploid stem-line comprising 50-55% and a haploid substem-line comprising 12-30% of the population. Estimation of cellular DNA contents by impulse cytoflowmetry and by Feulgen cytophotometry supported these observations. Because of their low diploid counts, these cell lines cannot be classified as diploid.     

Induction of apoptosis and CD10/neutral endopeptidase expression by jaspamide in HL-60 line cells

Jaspamide (jasplakinolide) is a natural peptide isolated from marine sponges of Jaspis species and has fungicidal and growth-inhibiting activities. We characterized the jasplakinolide-induced loss of viability by programmed cell death in the HL-60 human promyelocytic leukemia cell line and found that this process was accompanied by neutral endopeptidase (NEP)/CD10 expression on the surface of the apoptotic cells. HL-60 cells do not normally express detectable amounts of NEP/CD10 on their surface or intracytoplasmically, but upon jaspamide treatment, CD10 was synthesized de novo, its expression being inhibited by cycloheximide pretreatment. Once synthesized, NEP/CD10 interfered with the jasplakinolide signal delivered to HL-60 cells. Inhibition of NEP/CD10 by the NEP inhibitor phosphoramidon or by an anti-CD10 monoclonal antibody significantly increased apoptosis induction. The appearance of CD10 on the cell surface was blocked by preincubation of the cells with the monocytic/macrophage-differentiating agents vitamin D3 and phorbol 12-myristate 13-acetate, but not by the granulocytic differentiating agents retinoic acid or dimethyl sulfoxide. Moreover, in the promonocytic U937 and mature monocytic THP-1 cell lines, jaspamide induced apoptosis but not CD10 expression. In HL-60 cells, CD10 expression was partially but not totally blocked by the broad-spectrum caspase inhibitor benzyloxacarbonyl-Val-Ala-Asp-fluoromethylketone, indicating a connection between apoptosis induction and CD10 synthesis. Our findings suggest that the CD10 expression is related to the programmed cell death induction by jaspamide, and also with the process of granulocytic differentiation in HL-60 cells. Received 22 April 2002; received after revision 8 June 2002; accepted 10 June 2002     

Lysozymempfindlichkeit der SpeziesB. megaterium

Summary The 54 strains ofB. megaterium examined could be divided broadly into 2 groups on the basis of their lysozyme sensitivity. In most of the large-celled strains the cell walls were incompletely digested. Loss of turbidity on addition of lysozyme was comparatively slight and few free protoplasts were formed in the presence of saccharose. In contrast, the small-celled strains usually showed a marked drop in turbidity and complete protoplast formation could be obtained.These results suggest that, in some strains ofB. megaterium, particularly those possessing very large cells, certain substances are included in the cell walls which are not depolymerized by lysozyme.     

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.     

A synergistic interation between the teratogenic effect of trypan blue and dietary deficiency in the rat

Summary There was a increased incidence, compared to controls, of exencephaly and micropthalmia in the offspring of rats fed a vitamin D deficient diet and injected with trypan blue on day 9 of gestation. Oral vitamin D did not reverse the effect.Acknowledgments. The author wishes to thank Dr G. Lumb and Dr. B. Mawer for advice on diets, and Mrs de Silva for making the assays.     

Transfer of genetic information from T to B human lymphocytes during an immune response to herpes simplex virus]

Human blood lymphocytes carrying different allotypes were divided into B and T subpopulations and cultured in presence or in absence of ultraviolet inactivated Herpes Simplex Virus. Isolated B or T cells did not produce any antiherpetic activity. The B lymphocytes cultured in presence of 1 or 50% of the supernatant collected from virus exposed T cells synthesized on antiherpetic antibody with some allotypic markers of the T cell donor.     

Tumor suppressor role of protein 4.1B/DAL-1

Protein 4.1B/DAL-1 is a membrane skeletal protein that belongs to the protein 4.1 family. Protein 4.1B/DAL-1 is localized to sites of cell–cell contact and functions as an adapter protein, linking the plasma membrane to the cytoskeleton or associated cytoplasmic signaling effectors and facilitating their activities in various pathways. Protein 4.1B/DAL-1 is involved in various cytoskeleton-associated processes, such as cell motility and adhesion. Moreover, protein 4.1B/DAL-1 also plays a regulatory role in cell growth, differentiation, and the establishment of epithelial-like cell structures. Protein 4.1B/DAL-1 is normally expressed in multiple human tissues, but loss of its expression or prominent down-regulation of its expression is frequently observed in corresponding tumor tissues and tumor cell lines, suggesting that protein 4.1B/DAL-1 is involved in the molecular pathogenesis of these tumors and acts as a potential tumor suppressor. This review will focus on the structure of protein 4.1B/DAL-1, 4.1B/DAL-1-interacting molecules, 4.1B/DAL-1 inactivation and tumor progression, and anti-tumor activity of the 4.1B/DAL-1.     

Gene conversion in the chicken immunoglobulin locus: A paradigm of homologous recombination in higher eukaryotes

Gene conversion was first defined in yeast as a type of homologous recombination in which the donor sequence does not change. In chicken B cells, gene conversion builds the antigen receptor repertoire by introducing sequence diversity into the immunoglobulin genes. Immunoglobulin gene conversion continues at high frequency in an avian leukosis virus induced chicken B cell line. This cell line can be modified by homologous integration of transfected DNA constructs offering a model system for studying gene conversion in higher eukaryotes. In search for genes which might participate in chicken immunoglobulin gene conversion, we have identified chicken counterparts of the yeastRAD51, RAD52, andRAD54 genes. Disruption and overexpression of these genes in the chicken B cell line may clarify their role in gene conversion and gene targeting.     

Peritoneal macrophages from adjuvant arthritic rats enhance tumour cell growth in vitro

Summary Peritoneal macrophages from rats with adjuvant arthritis enhance the incorporation of³H-thymidine in 2 tumour cell lines in vitro. Maximal enhancement is found during the development of the secondary lesions, and it is suggested that the immunologic commitment of the macrophages could interfere with their regulation of tumour cell proliferation in vivo.Acknowledgments. The authors thank Drs P.F. Engel and M.P. Andreasen from the Finsen Institute, Copenhagen, for their gift of tumour cells and Ms E. Greve Petersen, Ms B. Hasselriis and Ms F. Norris for excellent technical assistance.     

Infection of cultured intestinal epithelial cells with severe acute respiratory syndrome coronavirus

To identify a model for the study of intestinal pathogenesis of severe acute respiratory syndrome (SARS) we tested the sensitivity of six human intestinal epithelial cell lines to infection with SARS coronavirus (SARS-CoV). In permissive cell lines, effects of SARS-CoV on cellular gene expression were analysed using high-density oligonucleotide arrays. Caco-2 and CL-14 cell lines were found to be highly permissive to SARS-CoV, due to the presence of angiotensin-converting enzyme 2 as a functional receptor. In both cell lines, SARS-CoV infection deregulated expression of cellular genes which may be important for the intestinal pathogenesis of SARS.Received 23 May 2004; received after revision 23 June 2004; accepted 25 June 2004