scholarly journals Production of Encecalin in Cell Cultures and Hairy Roots of Helianthella quinquenervis (Hook.) A. Gray

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3231
Author(s):  
J. Mabel Hernández-Altamirano ◽  
Irene F. Ugidos ◽  
Javier Palazón ◽  
Mercedes Bonfill ◽  
Penélope García-Angulo ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Hairy Roots ◽  
Cell Suspension Cultures ◽  
Gas Chromatography Mass Spectrometry ◽  
Wild Type ◽  
Transformed Roots ◽  
Organ Cultures ◽  
Chain Reaction ◽  
Biotechnological Production ◽  
Polymerase Chain

Plant cell and organ cultures of Helianthella quinquenervis, a medicinal plant whose roots are used by the Tarahumara Indians of Chihuahua, Mexico, to relieve several ailments, were established to identify and quantify some chromenes with biological activity, such as encecalin, and to evaluate their potential for biotechnological production. Gas chromatography–mass spectrometry (GC-MS) analysis corroborated the presence of quantifiable amounts of encecalin in H. quinquenervis cell cultures (callus and cell suspensions). In addition, hairy roots were obtained through three transformation protocols (prick, 45-s sonication and co-culture), using wild type Agrobacterium rhizogenes A4. After three months, cocultivation achieved the highest percentage of transformation (66%), and a comparable production (FW) of encecalin (110 μg/g) than the sonication assay (120 μg/g), both giving far higher yields than the prick assay (19 μg/g). Stable integration of rolC and aux1 genes in the transformed roots was confirmed by polymerase chain reaction (PCR). Hairy roots from cocultivation (six months-old) accumulated as much as 1086 μg/g (FW) of encecalin, over three times higher than the cell suspension cultures. The production of encecalin varied with growth kinetics, being higher at the stationary phase. This is the first report of encecalin production in hairy roots of H. quinquenervis, demonstrating the potential for a future biotechnological production of chromenes.

scholarly journals Multiplex PCR-based titration (MPBT) assay for determination of infectious titers of the three Sabin strains of live poliovirus vaccine

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Hasmik Manukyan ◽  
Elvira Rodionova ◽  
Tatiana Zagorodnyaya ◽  
Tsai-Lien Lin ◽  
Konstantin Chumakov ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Clinical Trials ◽  
Cell Cultures ◽  
Multiplex Polymerase Chain Reaction ◽  
Cytopathic Effect ◽  
Oral Poliovirus Vaccine ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Titration Assay ◽  
Serial Dilutions

Abstract Background Conventional assays to titrate polioviruses usually test serial dilutions inoculated into replicate cell cultures to determine a 50% cytopathic endpoint, a process that is both time-consuming and laborious. Such a method is still used to measure potency of live Oral Poliovirus Vaccine during vaccine development and production and in some clinical trials. However, the conventional method is not suited to identify and titrate virus in the large numbers of fecal samples generated during clinical trials. Determining titers of each of the three Sabin strains co-existing in Oral Poliovirus Vaccine presents an additional challenge. Results A new assay using quantitative multiplex polymerase chain reaction as an endpoint instead of cytopathic effect was developed to overcome these limitations. In the multiplex polymerase chain reaction-based titration assay, cell cultures were infected with serial dilutions of test samples, lysed after two-day incubation, and subjected to a quantitative multiplex one-step reverse-transcriptase polymerase chain reaction. All three serotypes of poliovirus were identified in single samples and titers calculated. The multiplex polymerase chain reaction-based titration assay was reproducible, robust and sensitive. Its lower limits of titration for three Sabin strains were 1–5 cell culture 50% infectious doses per ml. We prepared different combinations of three Sabin strains and compared titers obtained with conventional and multiplex polymerase chain reaction-based titration assays. Results of the two assays correlated well and showed similar results and sensitivity. Multiplex polymerase chain reaction-based titration assay was completed in two to 3 days instead of 10 days for the conventional assay. Conclusions The multiplex polymerase chain reaction-based titration (MPBT) is the first quantitative assay that identifies and titrates each of several different infectious viruses simultaneously in a mixture. It is suitable to identify and titrate polioviruses rapidly during the vaccine manufacturing process as a quality control test, in large clinical trials of vaccines, and for environmental surveillance of polioviruses. The MPBT assay can be automated for high-throughput implementation and applied for other viruses including those with no cytopathic effect.

scholarly journals Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (10) ◽  
pp. 5235-5243 ◽  
Author(s):  
D M Baronas-Lowell ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Wild Type ◽  
Diploid Strain ◽  
Chain Reaction ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ribosomal Subunits ◽  
Strong Homology ◽  
Polymerase Chain

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

A mutation in an exbD gene reduces tagetitoxin production by Pseudomonas syringae pv. tagetis

2006 ◽  
Vol 52 (11) ◽  
pp. 1027-1035 ◽  
Author(s):  
Hyesuk Kong ◽  
Cheryl D Patterson ◽  
Robin E Mitchell ◽  
Jeffrey S Buyer ◽  
M Catherine Aime ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Wild Type ◽  
Chain Reaction ◽  
Sequence Identity ◽  
Mutant Strains ◽  
Tonb System ◽  
Polymerase Chain ◽  
High Degree

A mutant of Pseudomonas syringae pv. tagetis EB037 with limited ability to produce tagetitoxin was isolated after transposon mutagenesis and the mutation was characterized. The mutation occurred in a gene with a high degree of sequence identity to exbD. exbD is contiguous with tonB and exbB upstream and with a gene for a TonB-dependent receptor downstream. Using reverse transcription – polymerase chain reaction with RNA from the wild-type and exbD mutant strains, we demonstrated that the mutation in exbD did not have a polar affect on the expression of downstream genes. The exbD mutant was able to grow well in conditions where iron is not freely available. Siderophore production by the exbD mutant was similar to that of the wild-type strain. We conclude that the mutation in exbD disrupts tagetitoxin production without compromising iron metabolism. The results indicate that tagetitoxin export by P. syringae pv. tagetis involves an efflux pump that requires a functional TonB system that is not essential for normal iron metabolism.Key words: Pseudomonas syringae pv. tagetis, Pseudomonas putida, tagetitoxin, exbD, exbB, tonB, TonB system, Helianthus annuus L.

Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae

1990 ◽  
Vol 10 (10) ◽  
pp. 5235-5243
Author(s):  
D M Baronas-Lowell ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Wild Type ◽  
Diploid Strain ◽  
Chain Reaction ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ribosomal Subunits ◽  
Strong Homology ◽  
Polymerase Chain

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

scholarly journals Interactions Between Antenatal Sulfadoxine-Pyrimethamine, Drug-Resistant Plasmodium falciparum Parasites, and Delivery Outcomes in Malawi

2020 ◽  
Vol 222 (4) ◽  
pp. 661-669 ◽  
Author(s):  
Steve M Taylor ◽  
Brandt Levitt ◽  
Betsy Freedman ◽  
Mwayiwawo Madanitsa ◽  
Kyaw-Lay Thwai ◽  
...  
Keyword(s):  
Birth Weight ◽  
Preventive Therapy ◽  
Sub Saharan Africa ◽  
Drug Resistant ◽  
Wild Type ◽  
Resistance Marker ◽  
Chain Reaction ◽  
Delivery Outcomes ◽  
Polymerase Chain ◽  
Sub Saharan

Abstract Background Sulfadoxine-pyrimethamine (SP) is used as intermittent preventive therapy in pregnancy (IPTp) for malaria in sub-Saharan Africa. The resistance marker dhps A581G has been associated with reduced IPTp-SP efficacy and enhanced morbidity in SP recipients. Methods We measured SP-resistance allele frequencies in Malawian women participating in a trial comparing IPTp with SP against intermittent screening by rapid diagnostic tests (ISTp). We genotyped polymerase chain reaction-detected parasites using deep sequencing of SP-resistance alleles. Results Among 125 placental infections, A581G-bearing parasites were associated with reduced birth weight (mean difference [MD], 252 g; 95% confidence interval [CI], 46–457; P = .017). Relative to ISTp, IPTp-SP was associated with higher birth weights in women with wild-type parasites (MD, 116 g; 95% CI, −40 to 272; P = .142) and lower birth weights in women with A581G-bearing parasites (MD, 192 g; 95% CI, −264 to 648; P = .385) (Pinteraction = .033). Similar associations were noted on gestational age (Pinteraction = .075). Amongst only IPTp-SP recipients, relative to women who last received SP > 4 weeks before delivery, recent SP receipt was associated with lower birth weight in women with wild-type parasites (MD, 118 g; 95% CI, −376 to 139; P = .361) and higher birth weight in women with A581G-bearing parasites (MD, 783 g; 95% CI, −20 to 1586; P = .054) (Pinteraction = .005). Conclusions The effectiveness in birth weight of IPTp-SP is compromised by A581G-bearing parasites, but there was no evidence that the adverse effects of these parasites are exacerbated by antenatal SP. ISRCTN Registry www.isrctn.com/ISRCTN69800930.

Clonal analysis of deletions on chromosome 20q and JAK2-V617F in MPD suggests that del20q acts independently and is not one of the predisposing mutations for JAK2-V617F

Blood ◽  
2009 ◽  
Vol 113 (9) ◽  
pp. 2022-2027 ◽  
Author(s):  
Franz X. Schaub ◽  
Roland Jäger ◽  
Renate Looser ◽  
Hui Hao-Shen ◽  
Sylvie Hermouet ◽  
...  
Keyword(s):  
Temporal Order ◽  
Jak2 V617f ◽  
Myeloproliferative Disorders ◽  
Genetic Alterations ◽  
Parental Origin ◽  
Wild Type ◽  
Chain Reaction ◽  
Colony Forming Units ◽  
Opposite Order ◽  
Polymerase Chain

We developed a real-time copy number polymerase chain reaction assay for deletions on chromosome 20q (del20q), screened peripheral blood granulocytes from 664 patients with myeloproliferative disorders, and identified 19 patients with del20q (2.9%), of which 14 (74%) were also positive for JAK2-V617F. To examine the temporal relationship between the occurrence of del20q and JAK2-V617F, we performed colony assays in methylcellulose, picked individual burst-forming units–erythroid (BFU-E) and colony-forming units–granulocyte (CFU-G) colonies, and genotyped each colony individually for del20q and JAK2-V617F. In 2 of 9 patients, we found that some colonies with del20q carried only wild-type JAK2, whereas other del20q colonies were JAK2-V617F positive, indicating that del20q occurred before the acquisition of JAK2-V617F. However, in colonies from 3 of 9 patients, we observed the opposite order of events. The lack of a strict temporal order of occurrence makes it doubtful that del20q represents a predisposing event for JAK2-V617F. In 2 patients with JAK2-V617F and 1 patient with MPL-W515L, microsatellite analysis revealed that del20q affected chromosomes of different parental origin and/or 9pLOH occurred at least twice. The fact that rare somatic events, such as del20q or 9pLOH, occurred more than once in subclones from the same patients suggests that the myeloproliferative disorder clone carries a predisposition to acquiring such genetic alterations.

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