ULTRASTRUCTURAL AND BIOCHEMICAL CHANGES IN BROWN FAT IN COLD-EXPOSED RATS

During the first 3 days of exposure of rats to 5°C, the nitrogen concentration of interscapular brown fat increased by 50% and remained at this elevated level for the duration of the 8-wk observation period, while the mass of tissue increased fourfold. The concentration of both DNA and RNA per unit nitrogen reached a maximum after 3 days, then declined; however, the total quantity of each continued to rise. The concentration of various respiratory enzymes decreased during the first few days and then increased, but at different rates. The morphological changes in mature brown fat cells during cold acclimation were observed to be: a reduction in fat droplet size during the first 3 days, followed by a gradual increase in size through 6 wk in the cold; a continual increase in the amount of intermitochondrial ground substance during the first 3 wk, with increased granularity and glycogen content after 1 wk; initial disappearance of glycogen between mitochondria, followed by the reappearance of a few isolated particles in the intermitochondrial ground substance after 1 wk in the cold; initial increase in the density of intramitochondrial matrix for the first 3–4 days, followed by a gradual return to the control density; loss in integrity of mitochondrial outer membranes during the first 4 days, followed by gradual but incomplete restoration; temporary loss of the dense material in lipid droplets during the first 24 hr, with return after 1 wk in the cold; and a 40% increase in mitochondrial diameter within 1 day, followed by a decrease in diameter within 1 wk to a constant value about 15% larger than the controls.     

Cloning and expression of soluble epoxide hydrolase from potato

Five cDNAs encoding a putative soluble epoxide hydrolase (sEH) from potato were isolated and characterized. The cDNAs contained open reading frames encoding 36 kDa polypeptides which were highly homologous to the carboxy terminal region of mammalian sEH. When one of the cDNAs was expressed in a baculovirus system a soluble 38 kDa protein with epoxide hydrolase activity was produced. The recombinant enzyme hydrolyzed a commonly used diagnostic substrate for the soluble form of mammalian EH. Inhibitor profiles of the recombinant potato and mammalian sEH were also similar. The expression of sEH in potato was found to be regulated by both developmental and environmental signals. Levels of mRNA for sEH were higher in meristematic tissue than in mature leaves. This mRNA was also observed to accumulate on wounding and application of exogenous methyl jasmonate.     

The action of caffeine on X-irradiated HeLa cells. VI. Damping of the structured age-survival function

Postirradiation treatment of synchronous HeLa S3 cultures with 4 mM caffeine until greater than or equal to 32 hr after mitotic collection, following exposure to 220-kV X rays at various times during interphase, severely damps the fluctuations in the age-survival curve. Not only does the dose-survival curve essentially lose its shoulder, as reported previously, but it becomes steeper and displays a virtually age-independent terminal slope (D0 congruent to 0.5 Gy). It becomes multicomponent, at least early in the cycle. The residual structure in the interphase age-survival curve, if any, appears to reflect mainly an age-dependent fluctuation in the size of a subpopulation of cells having marked sensitivity to X rays (D0 congruent to 0.25 Gy), though there might be small residual fluctuations in the size of the shoulder and the slope. Mitotic cells also respond to postirradiation treatment with caffeine; they yield a dose-survival curve whose slope is similar to that of the sensitive subpopulation seen in interphase. These findings indicate that most of the structure in the unperturbed age-survival function derives from repair of potentially lethal radiation damage.     

Oligonucleotide‐directed mutagenesis for precision gene editing

Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide‐directed mutagenesis (ODM), one of the many tools of Cibus’ Rapid Trait Development System ( RTDS ^?) technology, offers a rapid, precise and non‐transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide‐mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS , is reviewed.     

Caffeine-induced modulation of the lethal action of X rays on Chinese hamster V79 cells

Caffeine-mediated enhancement of the killing of V79 cells by 220-kV X rays at various times in the cell cycle was compared with that of HeLa cells by measuring (i) the dependence of cell survival on the duration of treatment with 5-10 mM caffeine, (ii) the effect of caffeine treatment on the X-ray dose-survival curve, and (iii) the loss of sensitivity to caffeine as a function of time after irradiation. The behavior of V79, while similar in many respects to that of HeLa (reported previously), differs in several ways. Caffeine treatment causes rapid killing immediately after irradiation irrespective of cell age, while HeLa is refractory in S phase and highly sensitive in G2. As with HeLa, the (multitarget) dose-survival curve parameters are reduced by caffeine treatment, but the age-dependent fluctuations in D0 are not eliminated as completely as with HeLa and the extrapolation number assumes values less than unity in the latter part of the cycle rather than in the early part. Loss of sensitivity to caffeine after irradiation early in the cycle appears to undergo a transient reversal in the middle of the cycle, a phenomenon not observed in HeLa.     

The action of caffeine on X-irradiated HeLa cells. VII. Evidence that caffeine enhances expression of potentially lethal radiation damage

HeLa cells irradiated with 2 Gy of 220-kV X rays suffer a 60-70% loss of colony-forming ability which is increased to 90% by postirradiation treatment with 10 mM caffeine for 6 hr. The detailed postirradiation patterns of cell death and sister-cell fusion in such cultures and in cultures in which the colony-forming ability was brought to about the same level by treatment with a larger (4 Gy) X-ray dose alone or by longer (48 hr) treatment with 10 mM caffeine alone were recorded by time-lapse cinemicrography. Because the patterns of cell death and fusion differ radically in irradiated and in caffeine-treated cultures, the response of the additional cells killed by the combined treatment can be identified as X-ray induced rather than caffeine induced. The appearance of cultures after several days of incubation confirms the similarity of the post-treatment patterns of proliferation in cultures suffering enhanced killing to those occurring in cultures treated with larger doses of X rays alone. It is concluded that X rays do not sensitize cells to caffeine, but rather that caffeine enhances the expression of potentially lethal radiation-induced damage.