Use of lysophosphatidylethanolamine, a natural lipid, to retard tomato leaf and fruit senescence

We studied the influence of lysophosphatidylethanolamine (LPE) on the pattern and rate of ethylene production and respiration of tomato ( Lycopersicon esculentum cv. H7155) leaflets and fruit. Leaflets that had been senescing on the plant showed a climacteric-like rise in ethylene production but not in respiration rate which decreased continuously with leaf age. Detached leaflets had a climacteric-like pattern in respiration whether they were incubated in complete darkness or in light. Detached leaflets incubated in the dark had higher rates of ethylene production and CO₂ evolution than did light-incubated leaves. There was no change in the pattern of ethylene production or CO₂ evolution as a result of LPE treatment. However, LPE-treated attached and detached leaflets had consistently lower rates of CO₂ evolution. The reduction in CO₂ evolution by LPE was most pronounced at the climacteric-like peak of the detached leaves. LPE-treated leaflets had a higher chlorophyll content and fresh weight and lower electrolyte leakage than the control. LPE-treated fruits had lower rates of ethylene and CO₂ production than did the control. LPE-treated fruits also had higher pericarp firmness and lower electrolyte leakage than the control. The results of the present study provide evidence that LPE is able to retard senescence of attached leaves and detached leaves and tomato fruits. Several recent studies suggest that lysolipids can act in a specific manner as metabolic regulators. Our results suggest a specific role of lysolipid LPE in aging and senescence     

Plant regeneration from nucllar tissues of Aegle marmelos through organogenesis

A protocol for organogenesis from nucellar explants excised from fertilized ovules of immature fruits of Aegle marmelos Corr. was developed. Adventitious buds were initiated on Murashige and Skoog's (MS) medium containing various combinations of 6-benzyladenine (BA), -naphthalene-acetic acid (NAA), 3-indoleacetic acid and gibberellic acid. Medium containing 4.4 m BA and 2.7 M NAA produced the maximum number of adventitious buds per explant. Shoots were elongated by transferring explants with shoot buds to medium with a low concentration of BA (0.44 M). Rooting of in vitro-regenerated shoots was obtained in half-strength MS medium with 4.9 M indole-3-butyric acid. This is the first report of plant regeneration from nucellar explants of A. marmelos.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - GA₃ gibberellic acid     

An index marker map of chromosome 9 provides strong evidence for positive interference.

An index marker map of chromosome 9 has been constructed using the Centre d'Etude du Polymorphisme Humain reference pedigrees. The map comprises 26 markers, with a maximum intermarker interval of 13.1 cM and only two intervals > 10 cM. Placement of all but one marker into the map was achieved with > 10,000:1 odds. The sex-equal length is 151 cM, with male length of 121 cM and female length of 185 cM. The map extends to within 2%-3% of physical length at the telomeres, and its coverage therefore is expected to be within 20-30 cM of full map length. The markers are all of the GT/CA repeat type and have average heterozygosity .77, with a range of .60-.89. The map shows both marked contraction of genetic distance relative to physical distance in the pericentromeric region and expansion in the telomeric regions. Genotypic data were carefully examined for errors by using the crossover routine of the program DATAMAN. Five new mutations were observed among 17,316 meiotic events examined. There were two double-crossover events occurring within an interval of 0-10 cM, and another eight were observed within an interval of 10-20 cM. Many of these could be due to additional mutational events in which one parental allele converted to the other by either gene conversion or random strand slippage. When there was no correction for these possible mutational events, the number of crossovers displayed by the maternal and paternal chromosomes was significantly different (P < .001) from that predicted by the Poisson distribution, which would be expected in the absence of interference. In addition, the observed crossover distribution for paternally derived chromosomes was similar to that predicted from cytogenetic chiasma frequency observations. In all, the data strongly support the occurrence of strong positive interference on human chromosome 9 and suggest that flanking markers at an interval of < or = 20 cM are generally sufficient for disease gene inheritance predictions in presymptomatic genetic counseling by linkage analysis.     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 10⁶ cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 10⁸ TCID₅₀/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

Identification of new mutations in the Cu/Zn superoxide dismutase gene of patients with familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. Although most cases of ALS are sporadic, approximately 10% are inherited as an autosomal dominant trait. Mutations in the Cu/Zn superoxide dismutase gene (SOD 1) are responsible for a fraction of familial ALS (FALS). Screening our FALS kindreds by SSCP, we have identified mutations in 15 families, of which 9 have not been previously reported. Two of the new mutations alter amino acids that have never been implicated in FALS. One of them affects a highly conserved amino acid involved in dimer contact, and the other one affects the active-site loop of the enzyme. These two mutations reduce significantly SOD 1 enzyme activity in lymphoblasts. Our results suggest that SOD 1 mutations are responsible for > or = 13% of FALS cases.     

A comparison of some pharmacological actions of prostaglandin E1, 6-oxo-PGE1 and PGI2

Some pharmacological actions of prostaglandin E1 (PGE1), 6-oxo-PGE1 and PGI2 have been studied. 6-oxo-PGE1 and PGE1 relaxed guinea-pig tracheal muscle in vitro and increased nasal patency in normal volunteers and in subjects with vasomotor rhinitis whereas PGI2 produced opposite effects. All three compounds produced bronchodilatation in the anaesthetised guinea-pig and relaxed human respiratory tract muscle in vitro. PGI2 was several times more potent than either 6-oxo-PGE1 or PGE1 against ADP-induced aggregation of human and baboon platelets in vitro. Intravenous 6-oxo-PGE1 in the baboon caused an ex vivo inhibition of platelet aggregation, but the EC50 was 7.7 times that of PGI2. As a vasodepressor in the baboon 6-oxo-PGE1 and PGI2 were equipotent. Thus with the exception of the vasodepressor effect, the actions of 6-oxo-PGE1 qualitatively and quantitatively resembled those of the structurally related PGE1 rather than those of PGI2.