Protein synthesis and loss of viability in rye embryos. The lability of transferase enzymes during senscence.
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ABSTRACT:
SUBMITTER: Roberts BE
PROVIDER: S-EPMC1165842 | biostudies-other | 1973 Nov
REPOSITORIES: biostudies-other
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Project description:A decline in protein synthesis and slow germination accompanies loss of viability in embryos of rye (Secale cereale L.) grains. Associated with this, incorporation of precursors into all the major classes of RNA is decreased and the processing of precursor rRNA to 25S and 18S RNA is retarded. Embryos that just reach 0% viability still synthesize some low-molecular-weight non-nucleolar material, although they do not synthesize protein. It is suggested that early-synthesized RNA could play a major part in determining the extent of protein synthesis at early germination, and thereby regulate the rate at which germination can proceed.
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Project description:A study was made of the integrity of some components of the protein-synthesizing system from viable and non-viable embryos of rye grains. In comparison with viable-embryo components both post-ribosomal supernatant and ribosomal fractions from non-viable embryos are impaired, for neither will fully support polyphenylalanine synthesis in poly(U)-directed cell-free systems. The lesion in the supernatant lies in components other than the tRNA or the aminoacyl-tRNA synthetase, for these are as functional as those present in the fully active cell-free systems from viable embryos. The ribosomes of embryos of lowered viability show considerable fragmentation and degradation of both 18S and 25S rRNA. This breakdown does not, however, account for the complete lack of polypeptide synthesis in the poly(U)-directed non-viable-embryo system, for if provided with viable-embryo supernatant, non-viable-embryo ribosomes will sustain 60% of the viable-embryo ribosome activity. A lesion in non-viable-embryo supernatant has been located in the binding of the aminoacyl-tRNA to the ribosome. The impaired components in both supernatant and ribosomes in systems in vitro may reflect the site of faults in protein synthesis in vivo in the early hours of germination. The development of these lesions during grain storage could contribute to senescence and loss of viability in the embryos of rye.
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Project description:Incorporation studies with radioactive precursors showed that synthesis of protein and RNA is initiated in germinating embryos of rye within the first hour of imbibition of water. By polyacrylamide-gel fractionations of radioactive nucleic acid components, the appearance of products of transcription of the genome was shown to follow the sequence: heterogeneous (ribonuclease-sensitive) RNA, 4S and 5S RNA by 20min, 31S and 25S rRNA by 40min, and 18S RNA by 60min. "Fingerprint' analysis of T1-ribonuclease digests show that all the large oligonucleotides present in 25S and 18S RNA are present in the 31S species, indicating that 31S RNA is the precursor rRNA molecule to both 25S and 18S RNA. The importance of these early RNA syntheses and in particular the possible template function of the heterogeneous RNA is discussed in relation to the concept of long-lived mRNA and the coding for protein synthesis in the first hours of germination.
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Project description:In vitro fertilisation (IVF) and related technologies are arguably the most challenging of all cell culture applications. The starting material is a single cell from which one aims to produce an embryo capable of establishing a pregnancy eventually leading to a live birth. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. To reduce the risk of cellular stress, we have developed a totally enclosed system of interlinked isolator-based workstations designed to maintain oocytes and embryos in a physiological environment throughout the IVF process. Comparison of clinical and laboratory data before and after the introduction of the new system revealed that significantly more embryos developed to the blastocyst stage in the enclosed isolator-based system compared with conventional open-fronted laminar flow hoods. Moreover, blastocysts produced in the isolator-based system contained significantly more cells and their development was accelerated. Consistent with this, the introduction of the enclosed system was accompanied by a significant increase in the clinical pregnancy rate and in the proportion of embryos implanting following transfer to the uterus. The data indicate that protection from ambient conditions promotes improved development of human embryos. Importantly, we found that it was entirely feasible to conduct all IVF-related procedures in the isolator-based workstations.
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Project description:Protein film voltammetry (PFV) is used to interrogate the behavior of a variety of bacterial and mitochondrial His/Met-ligated cytochromes c. While analogous studies upon alkanethiol-modified gold electrodes reveal the anticipated Fe(II/III) couple only, PFV using pyrolytic graphite edge (PGE) electrodes demonstrates the presence of a lower-potential form of each of the cyts c studied, with a potential of approximately -100 mV (vs hydrogen). The generation of the novel, lower-potential state is shown to arise specifically from the interaction with the PGE electrode. Simultaneously, the typical Fe(II/III) couple can be observed. PFV of a series of wild-type cytochromes and mutants in the Met-donating loop show that the lower-potential state is highly similar between proteins from Pseudomonas aeruginosa (PA), Hydrogenobacter thermophilus (HT), and horse heart. The generation of the lower-potential form correlates inversely with the stability of the Met-Fe interaction for each of the cytochromes. Comparison with chemically unfolded cyts c indicates that the lower-potential forms detected here are unique, and this distinct state is ascribed to the loss of the Met ligand. Thus, PGE is demonstrated to be a non-innocent electrode surface in PFV studies of His/Met-ligated cytochromes c.
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Project description:Main conclusionModulation of the gaseous environment using oxygen absorbers and/or silica gel shows potential for enhancing seed longevity through trapping toxic volatiles emitted by seeds during artificial ageing. Volatile profiling using non-invasive gas chromatography-mass spectrometry provides insight into the specific processes occurring during seed ageing. Production of alcohols, aldehydes and ketones, derived from processes such as alcoholic fermentation, lipid peroxidation and Maillard reactions, are known to be dependent on storage temperature and relative humidity, but little is known about the potential modulating role of the gaseous environment, which also affects seed lifespan, on volatile production. Seeds of Lolium perenne (Poaceae), Agrostemma githago (Caryophyllaceae) and Pisum sativum (Fabaceae) were aged under normal atmospheric oxygen conditions and in sealed vials containing either oxygen absorbers, oxygen absorbers and silica gel (equilibrated at 60% RH), or silica gel alone. Seeds of A. githago that were aged in the absence of oxygen maintained higher viability and produced fewer volatiles than seeds aged in air. In addition, seeds of A. githago and L. perenne aged in the presence of silica gel were longer lived than those aged without silica, with no effect on seed moisture content or oxygen concentration in the storage containers, but with silica gel acting as a volatile trap. These results indicate that the use of inexpensive oxygen absorbers and silica gel could improve seed longevity in storage for some species and suggests a potential, and previously unidentified, role for silica gel in ultra-dry storage.
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Project description:The stage differentiation from trophozoite to cyst (i.e., encystation) is an essential step for Giardia to survive outside its human host and spread the infection via the fecal-oral route. We have previously shown that Giardia expresses glucosylceramide transferase 1 (GlcT1) enzyme, the activity of which is elevated during encystation. We have also reported that blocking the activity of gGlcT1 interferes with the biogenesis of encystation-specific vesicles (ESVs) and cyst viability in Giardia. To further understand the role of this enzyme and how it regulates encystation, we overexpressed, knocked down, and rescued the giardial GlcT1 (gGlcT1) gene and measured its enzymatic activity in live parasites as well as in isolated membrane fractions using NBD-ceramide and UDP-glucose or UDP-galactose. We observed that gGlcT1 is able to catalyze the synthesis of both glucosylceramide (GlcCer) and galactosylceramide (GalCer), however the synthesis of GalCer is 2-3 fold higher than of GlcCer. Although both activities follow Michaelis-Menten kinetics, the bindings of UDP-glucose and UDP-galactose with the enzyme appear to be non-competitive and independent of each other. The modulation of gGlcT1 synthesis concomitantly influenced the expression cyst-wall protein (CWP) and overall encystation. We propose that gGlcT1 is a unique enzyme and that Giardia uses this enzyme to synthesize both GlcCer and GalCer to facilitate the process of encystation/cyst production.
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Project description:Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Moreover, autophagy is essential for preimplantation development in mammals. Here we show that autophagy is also important for reprogramming in somatic cell nuclear transfer (SCNT). Our data indicate that unlike fertilized oocytes, autophagy is not triggered in SCNT embryos during 6 hours of activation. Mechanistically, the inhibited autophagic induction during SCNT activation is due to the cytochalasin B (CB) caused depolymerization of actin filaments. In this study, we induced autophagy during SCNT activation by rapamycin and pp242, which could restore the expected level of autophagy and significantly enhance the development of SCNT embryos to the blastocyst stage when compared with the control (68.5% and 68.7% vs. 41.5%, P < 0.05). Furthermore, the treatment of rapamycin and pp242 accelerates active DNA demethylation indicated by the conversion of 5 mC to 5 hmC, and treatment of rapamycin improves degradation of maternal mRNA as well. Thus, our findings reveal that autophagy is important for development of SCNT embryos and inhibited autophagic induction during SCNT activation might be one of the serious causes of low efficiency of SCNT.
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