Nonproducer Research Articles

Rapid PCR Method for the Selection of 1,3-Pentadiene Non-Producing Debaryomyces hansenii Yeast Strains.

To prevent microbial growth and its consequences, preservatives such as sorbic acid or its salts, commonly known as sorbates, are added to foods. However, some moulds and yeasts are capable of decarboxylating sorbates and producing 1,3-pentadiene. This is a volatile compound with an unpleasant “petroleum-like “odour, which causes consumer rejection of the contaminated products. In this work, we studied the production of 1,3-pentadiene in 91 strains of the yeast Debaryomyces hansenii, and we found that nearly 96% were able to produce this compound. The sequence of the FDC1Dh gene was analysed showing differences between 1,3-pentadiene producer (P) and non-producer (NP) strains. A specific PCR assay with degenerated primers based on the gene sequence was developed to discern NP and P strains. It was tested on D. hansenii strains and on some physiologically related species frequently isolated from foods, such as D. fabrii, D. subglobosus and Meyerozyma guillermondii. This method could be applied for the selection of NP D. hansenii strains, useful in biotechnological food production and as a biocontrol agent.

Experimental Biofilm-Related Candida Infections

We investigated the pathogenic role of biofilm and the therapeutic efficacy of anidulafungin in experimental infections of the wax moth Galleria mellonella by Candida albicans clinical strains. On the basis of the in vitro propensity to form biofilm, five biofilm-producer (BP) and four nonproducer (NP) C. albicans clinical strains were used in this study. For each strain, we assessed the virulence by infecting G. mellonella larvae and observing survival. Anidulafungin was administered 2 h after yeast inoculum at 0.6 µg, according to the therapeutic dose recommended for humans. Biofilm-forming ability highly influenced the larva-killing rate. A significant (p < 0.0001) survival decrease was observed in the BP group, with 80% of the infected larvae dying within 72 h. NP isolates did not reach the same killing rate, even at the end of experiments (216 h). Larval survival was enhanced (p < 0.0001) by anidulafungin administration in both groups. Survival rate at 72 h was similar in both groups (BP 78.5% and NP 87.5%); whereas there were still differences at the end of the experiments, with a higher survival in the NP group (75 vs 48%). Our data confirm the pathogenic role of biofilm in C. albicans infections. Its importance was further enhanced by a lack of contribution from extracellular enzymes, detected in both NP and BP strains. In addition, we demonstrated anidulafungin efficacy in treating biofilm-related invasive candidiasis.

Equol production changes over time in pre-menopausal women

Equol (EQ) is a metabolite produced by gut bacteria through the chemical reduction of the soya isoflavone daidzein (DE), but only by 30-60% of the population. EQ is believed to provide benefits derived from soya intake and its production is widely viewed as a relatively stable phenomenon. In a randomised, cross-over intervention with soya foods, seventy-nine pre-menopausal women were challenged with a high-soya and a low-soya diet each for 6 months, separated by a 1-month washout period. Overnight urine was collected at three time points during each diet period and analysed for DE and EQ by liquid chromatography tandem MS. Remaining an EQ producer (EP) or non-producer (NP) or changing towards an EP or NP was assessed using an EQ:DE ratio of ≥0·018 combined with a DE threshold of ≥2 nmol/mg creatinine as a cut-off point. We observed 19 and 24% EP during the low-soya and high-soya diet periods, respectively, and found that 6-11% of our subjects changed EQ status 'within' each study period (on an average of 1·2 times), while 16% changed 'between' the two diet periods. The present finding challenges the widely held conviction that EQ production within an individual remains stable over time. The precise factors contributing to changes in EQ status, however, remain elusive and warrant further investigation.

OKIO-Transformed Cell Lines: Viral Component Generation and Tumorigenicity

Stable nonproducer (NP) cell lines transformed by the avian acute leukemia virus OK10 were established. All 13 studied NP quail cell lines released into the culture medium noninfectious, mostly reverse-transcriptase-negative particles containing the usual gag proteins. Infectious, transforming OK10 virus pseudotypes could be recovered by rescue with helper virus. p200, the putative transforming protein of OK10, was identified in in vitro translates of RNA from the noninfectious particles and in immunoprecipitates of cell extracts of the NP clones analyzed. Two NP clones, the reverse-transcriptase-negative B5 and -positive 9C cell lines, displayed striking differences in in vivo tumorigenicity. Although B5 induced no tumors in quails, 9C caused multiple tumors and cells derived from several tumors could be passaged in vitro. At no time during the in vivo passage or during more than 1 year of in vitro culture have the particles released by 9C cells acquired infectious properties. Possible reasons for the observed differences in tumorigenicity between the OK10 virus-transformed 9C and B5 NP cell lines are discussed.

Size and genetic content of virus-specific RNA in myeloblasts transformed by Avian Myeloblastosis Virus (AMV)

Radiolabeled complementary DNA probes representing sequences specific for avian myeloblastosis virus (AMV) and sequences of different regions of avian leukosis virus (ALV) genome were used to analyze the gene content and size distribution of viral-specific RNA in AMV-transformed myeloblasts. The producer myeloblasts contained 8–10 times more copies per cell of virus-specific RNA than those of nonproducer (NP) myeloblasts. In the producer myeloblast, 35 S, 34 S, and 21 S virus-specific RNAs were detected. The 35 S RNA contained gag, pol, env, and c sequences; representing both the genomic and the messenger RNA of the helper virus. The 34 S RNA contained gag, pol, AMV, and c sequences, representing the genomic and the messenger RNA of AMV. The 21 S RNA of producer cells contained two different species: one contained env and c sequences and represented the glycoprotein messenger RNA of the helper virus; another contained AMV and c sequences and represented the subgenomic messenger RNA of AMV. In NP cells, two virus-specific RNAs were found; a 34 S RNA containing gag, pol, AMV, and c sequences and a 21 S RNA containing AMV and c sequences. Both RNAs hybridized to a probe specific for 5′ sequences of ALV. About 15% of pol sequences of virus-specific RNA in NP cells were found to be deleted.

Development of virus non-producer lymphosarcomas in pet cats exposed to FeLv.

Naturally occurring oncoviruses of several species are transmitted contagiously and cause lymphosarcoma (LSA) or leukaemia in their hosts. All naturally occurring oncoviruses replicate in vivo in the tumours they induce or, as with bovine leukaemia virus, can be isolated from tumour cells grown in short-term cell culture. However, we have shown that feline leukemia virus (FeLV) is not present in a significant minority of pet cats that develop LSA. Unlike experimentally induced virus-negative leukaemias and sarcomas of other species, LSA cells from FeLV-negative LSA cats lack any FeLV proteins, including p15 or p12, and complete functional copies of FeLV provirus and thus do not produce FeLV when grown in cell culture. Thus, except for FeLV, the naturally occurring animal leukaemogenic oncoviruses seem to induce only virus-producing lymphoid tumours. Our earlier findings prompted a study to determine the frequency of occurrence of FeLV non-producer (NP) LSA in pet cats and whether NP LSAs develop in cats exposed to FeLV. We report here epidemiological data which indicate that development of NP LSAs in pet cats is associated with exposure to FeLV and suggest that FeLV may be the aetiological agent for FeLV NP feline LSAs. Thus, feline NP LSAs may be suitable for studying the potential viral aetiology and mechanism of leukaemogenesis of human lymphoid tumours in which no oncoviruses have, as yet, been proved to cause the disease.

"Nonproductive" Mouse Sarcoma Virus Transformed Gorilla Cells which Contain Noninfectious Type C Virus Particles

Gorilla spleen cells can be readily transformed morphologically in vitro by Kirsten murine sarcoma virus (Ki-MSV) and the MSV pseudotype of baboon placenta virus-MSV (BP). Gorilla spleen cells transformed in vitro by the MSV (BP) were found to be virus producers; whereas Ki-MSV transformed gorilla spleen cells were found to be nonproducer (NP). These NP cells were obtained by simply infecting gorilla cells with Ki-MSV and subculturing them. These morphologically altered NP cells did not produce infectious virus or murine leukemia virus gs antigen, but contained a rescuable MSV genome and also noninfectious type-C virus particles.

Defectiveness of avian myelocytomatosis virus MC29: Isolation of long-term nonproducer cultures and analysis of virus-specific polypeptide synthesis

Nonproducer (NP) clones of chicken and quail cells transformed by avian myelocytomatosis virus MC29-A were isolated in focus and agar colony assays. Several quail MC29 NP clones were developed into long-term cultures. They have been kept in continuous culture for 6 months and about 35 passages. They do not release virus particles detectable by [ 3H]uridine incorporation or reverse transcriptase assay. Rescue of transforming virus is possible at any time by superinfection of the NP cell clones with avian leukosis viruses such as Rous associated virus type 1 or ring-necked pheasant virus (RPV). [ 35S]Methionine pulse-labeled protein extracts of NP and of superinfected MC29 transformed cell cultures were analyzed by immune precipitation and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In NP cells the common precursor polypeptide for the structural proteins of avian RNA tumor viruses (pr76) is not synthesized. Instead, a major polypeptide with a molecular weight of 110,000–120,000 was observed (MC29–110K). In superinfected cells, both MC29–110K and pr76 are synthesized. The MC29–110K polypeptide was precipitated by an antiserum against whole virus (PR RSV-B) as well as by a monospecific anti-p27 serum. It was not precipitated by an anti-glycoprotein serum. Pulse-chase experiments showed that the MC29–110K polypeptide turned over at a rate comparable to that of pr76. However, none of the major structural proteins (p27, p19, p15) could be detected after the chase. Competition radioimmune assays demonstrated that protein extracts of NP MC29 cells contain inhibitory activity for precipitation of p19 and p27, but not for p15.

Characterization of non‐producer human cells induced by kirsten sarcoma virus

Non-producer (NP) human cells induced by the Kirsten sarcoma virus were characterized. These morphologically altered NP cells produced neither infectious virus nor complement-fixing antigens of the murine sarcoma-leukemia virus complex. The NP cells did not release RNA-dependent DNA polymerase and type-C virus particles with a density of approximately 1.15 g/ml in sucrose gradients by 3H-uridine labelling. The NP cells produced tumors when transplanted subcutaneously into athymic nude mice. The tumor cells re-established in culture resembled the orginal NP cells, were confirmed as human cells by karyological analysis and were also found to be "non-producer". The sarcoma virus genome in NP cells could be rescued not only by co-cultivation with "helper virus"-releasing cells but also by superinfection with helper type-C viruses. Murine (Rauscher, Ki-MuLV, AT-124 and two other xenotropic viruses), feline, RD-114 and Simian (woolly monkey and baboon) type-C viruses possessed the ability to rescue the sarcoma genome from NP cells but not AKR leukemia virus. In addition, the feline leukemia virus titer obtained by the rescuing technique in NP cells was the same as those obtained in feline embryo and NP cells by CF induction assay.

MUTANTS OF NONPRODUCER CELL LINES TRANSFORMED BY MURINE SARCOMA VIRUS

BALB/3T3 cells transformed by the Kirsten sarcoma virus (nonvirus producer BALB/3T3 cells) and mutant cell lines derived therefrom by treatment with bromodeoxyuridine (BrdU) were analyzed for expression of virus-specific RNA using single-stranded DNA transcripts of Rauscher leukemia virus (RLV), a virus activated in one of the cell lines (58-2T), and Ki-SV-specific DNA transcript; the latter transcript after removal of all sequences cross-reactive with RLV RNA. The Rauscher virus DNA detected multiple copies of viral RNA in virus-producing cells ( approximately 2.5 x 10(3)/cell) whether infected with RLV or activated to produce virus with BrdU. Nonproducer (NP) cells and normal BALB cells showed small numbers of RNA genomes (70-250/cell) and only partial saturation of the transcript. The intracellular RNA sedimented at 35S (main peak) with a variable minor peak at 20S with the exception of one mutant cell, M-43-2 (main peak at 26-27S). The 58-2T transcript reacted preferentially in NP cells and their derivatives with biphasic kinetics suggesting the possibility of sequences specific for the original transforming virus. The size of Ki-SV specific sequences were 30S in mutant cells whether or not complete virus was being produced and independent of in vivo transplantability.

Mutants of nonproducer cell lines transformed by murine sarcoma viruses. I. Induction, isolation, particle production, and tumorigenicity.

A variety of cell mutants were obtained by a single 5'-bromodeoxyuridine (BrdU) treatment of an nonproducer (NP) cell line transformed by the Kirsten strain of murine sarcoma virus (Ki-MSV). Isolation procedures of these cell See PDF for Structure mutants are described. The cell mutants obtained were classified by tumorigenic potential and shedding of Type C virus particles. The cell mutants were classified into four groups: (A) tumorigenic, without particles; (B) tumorigenic, with Type C particles; (C) nontumorigenic, without particles; and (D) nontumorigenic, with Type C particles. The tumorigenic cell lines showed variability in morphology with both flat and typical transformed appearing cell lines showing equal transplantability.

Mutants of nonproducer cell lines transformed by murine sarcoma virus. II. Relationship of tumorigenicity to presence of viral markers and rescuable sarcoma genome.

Tumorigenic and nontumorigenic mutants induced by a single 5'-bromodeoxyuridine (BrdU) treatment of a nonproducer (NP) tumorigenic cell line were isolated and characterized. Among the cloned derivatives were examples of virus-free and sarcoma virus-producing cell lines. Oncogenicity did not correlate with production of virus or ease of rescue of the sarcoma genome. All lines, including nononcogenic derivatives, retained the sarcoma genome. Phenotypic reversion of some cell mutants was observed after in vivo inoculation or long term in vitro cultivation. The M-50T cell line, obtained from a tumor induced by M-50 cells, had a sarcoma genome rescuable by direct superinfection; this was only achieved with parental M-50 cells by a cell fusion rescue technique. The M-43-2T cell, obtained from a single small static tumor induced by otherwise nononcogenic M-43-2 cells, shed sarcoma virus and became tumorigenic. M-58-4-48 became tumorigenic after passage 48 of the M-58-4 line, which was originally nontumorigenic. These observations of phenotypic reversion demonstrate that the presence of the sarcoma gene in cells is an essential but not sufficient condition of tumorigenesis.