Sarcomere matrix of striated muscle: in vivo phosphorylation of titin and nebulin in mouse diaphragm muscle

Titin and nebulin are two major protein components of a cytoskeletal matrix that coexists with thick and thin filaments within the sarcomere of a wide range of striated muscles. Purified titin and nebulin from mouse diaphragm muscle are similar in size, in relative abundance, and in amino acid composition to analogous proteins from other mammals or avians. Phosphate analysis of these nucleic-acid-free proteins indicated that both proteins contain substantial amounts of protein-bound phosphate: about 12 mol of phosphate per mole of titin subunit and 11 mol of phosphate per mole of nebulin subunit. Incubation of intact, excised mouse diaphragm with radioactive inorganic phosphate resulted in significant incorporation of radiophosphate into titin and nebulin. The identification of titin and nebulin phosphorylation was facilitated by a simple salt fractionation and nuclease digestion procedure that effectively separated titin and nebulin from radiolabeled nucleic acids. Such in vivo phosphorylation studies indicated that approximately 2 mol of phosphate per titin subunit and 5 to 7 mol of phosphate per nebulin subunit were incorporated within 5 h of incubation. The incorporation nearly doubled when the beta-adrenergic agonist, isoproterenol, or a phosphodiesterase inhibitor, theophylline, was present in the medium. For both proteins, phosphorylation occurred mainly on serine residues. Nebulin also appears to possess a smaller number of threonine sites. Taken together, our data indicate that a small proportion (20 to 40%) of the steady-state titin phosphates are rapidly turning over. In contrast, most of the nebulin phosphates (50 to 100%) are readily exchanged. The modulation of turnover by external stimuli that increase cytosolic cAMP raises the possibility that at least a portion of the multiple phosphorylation sites of titin and nebulin may be involved in the functional regulation of the sarcomere matrix.     

A Mutant of Arabidopsis thaliana that Exhibits Chlorosis in Air but Not in Atmospheres Enriched in CO(2)

A mutant of Arabidopsis thaliana (L.) Heynh. which requires a high concentration (2% by volume) of atmospheric CO₂ for growth has been isolated. Unlike previous mutants of this type, this line does not have any apparent defect in photosynthetic CO₂-fixation, photorespiration, or photosynthetic electron transport. The mutant is abnormally susceptible to pigment bleaching in air but not in 2% CO₂. The presence of normal or above-normal levels of antioxidants, carotenoids, and enzymes involved in reactive oxygen detoxification suggests that the mutant is equipped to detoxify activated oxygen species. Although it was not possible to establish a biochemical basis for the lesion, the properties of the mutant suggest the existence of a previously unidentified role for CO₂.     

The role of cytochrome b5 in delta 12 desaturation of oleic acid by microsomes of safflower (Carthamus tinctorius L.)

The electron donors for the membrane-bound fatty acid desaturases of higher plants have not previously been identified. In order to assess the participation of cytochrome b5 in microsomal fatty acid desaturation, the cytoplasmic domain of microsomal cytochrome b5 was purified from Brassica oleracea, and murine polyclonal antibodies were prepared. The IgG fraction from ascites fluid inhibited 62% of NADH-dependent cytochrome c reduction in safflower (Carthamus tinctorius L.) microsomes. These antibodies also blocked desaturation of oleic acid to linoleic acid in lipids of C. tinctorius microsomes by 93%, suggesting that cytochrome b5 is the electron donor for the delta 12 desaturase.     

LKB1, Salt-Inducible Kinases,and MEF2C Are Linked Dependencies in Acute Myeloid Leukemia

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TOM, a new aromatic degradative plasmid from Burkholderia (Pseudomonas) cepacia G4.

Burkholderia (Pseudomonas) cepacia PR1(23) has been shown to constitutively express to toluene catabolic pathway distinguished by a unique toluene ortho-monooxygenase (Tom). This strain has also been shown to contain two extrachromosomal elements of < 70 and > 100 kb. A derivative strain cured of the largest plasmid, PR1(23) Cure, was unable to grow on phenol or toluene as the sole source of carbon and energy, which requires expression of the Tom pathway. Transfer of the larger plasmid from strain G4 (the parent strain inducible for Tom) enabled PR1(23) Cure to grow on toluene or phenol via inducible Tom pathway expression. Conjugal transfer of TOM23c from PR1(23) to an antibiotic-resistant derivative of PR1(23) Cure enabled the transconjugant to grow with either phenol or toluene as the sole source of carbon and energy through constitutive expression of the Tom pathway. A cloned 11.2-kb EcoRI restriction fragment of TOM23c resulted in the expression of both Tom and catechol 2,3-dioxygenase in Escherichia coli, as evidenced by its ability to oxidize trichloroethylene, toluene, m-cresol, o-cresol, phenol, and catechol. The largest resident plasmid of PR1 was identified as the source of these genes by DNA hybridization. These results indicate that the genes which encode Tom and catechol 2,3-dioxygenase are located on TOM, an approximately 108-kb degradative plasmid of B. cepacia G4.     

Enhanced thermal tolerance in a mutant of Arabidopsis deficient in palmitic Acid unsaturation

A mutant of Arabidopsis thaliana, deficient in the activity of a chloroplast ω9 fatty acid desaturase, accumulates high amounts of palmitic acid (16:0), and exhibits an overall reduction in the level of unsaturation of chloroplast lipids. Under standard conditions the altered membrane lipid composition had only minor effects on growth rate of the mutant, net photosynthetic CO₂ fixation, photosynthetic electron transport, or chloroplast ultrastructure. Similarly, fluorescence polarization measurements indicated that the fluidity of the membranes was not significantly different in the mutant and the wild type. However, at temperatures above 28°C, the mutant grew more rapidly than the wild type suggesting that the altered fatty acid composition enhanced the thermal tolerance of the mutant. Similarly, the chloroplast membranes of the mutant were more resistant than wild type to thermal inactivation of photosynthetic electron transport. These observations lend support to previous suggestions that chloroplast membrane lipid composition may be an important component of the thermal acclimation response observed in many plant species which are photosynthetically active during periods of seasonally variable temperature extremes.     

Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C_16:1 and C_18:1 lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.     

Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity

Mutants of Arabidopsis thaliana deficient in plastid glycerol-3-phosphate acyltransferase activity have altered chloroplast membrane lipid composition. This caused an increase in the number of regions of appressed membrane per chloroplast and a decrease in the average number of thylakoid membranes in the appressed regions. The net effect was a significant decrease in the ratio of appressed to nonappressed membranes. A comparison of 77 K fluorescence emission spectra of thylakoid membranes from the mutant and wild type indicated that the ultrastructural changes were associated with an altered distribution of excitation energy transfer from antenna chlorophyll to photosystem II and photosystem I in the mutant. The changes in leaf lipid composition did not significantly affect growth or development of the mutant under standard conditions. However, at temperatures above 28°C the mutant grew slightly more rapidly than the wild type, and measurements of temperature-induced fluorescence yield enhancement suggested an increased thermal stability of the photosynthetic apparatus of the mutant. These effects are consistent with other evidence suggesting that membrane lipid composition is an important determinant of chloroplast structure but has relatively minor direct effects on the function of the membrane proteins associated with photosynthetic electron transport.     

A mutant of Arabidopsis deficient in the chloroplast 16:1/18:1 desaturase

Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 18-carbon and 16-carbon polyunsaturated fatty acids and increased levels of the 18:1 and cis-16:1 precursors due to a single nuclear mutation at a locus designated fadC. Analysis of the fatty acid compositions of individual lipids and the kinetics of lipid labeling with [¹⁴C]acetate in vivo indicate that the mutant lacks activity of the chloroplast glycerolipid ω-6 desaturase. As a result, lipids synthesized by the prokaryotic pathway are not desaturated further than 18:1 and 16:1. Lipids derived from the eukaryotic pathway are desaturated—presumably by the endoplasmic reticulum 18:1 phosphatidylcholine desaturase. However, an increase in the level of 18:1 on all the phospholipids derived from the eukaryotic pathway in leaves of the mutant suggests that the mutation does exert an effect on the composition of extrachloroplast membranes. Synthesis of monogalactosyldiacylglycerol (MGD) by the prokaryotic pathway is reduced 30 to 35% in the mutant and there is a corresponding increase in MGD synthesis by the eukaryotic pathway. This shift in metabolism which results in a more unsaturated MGD pool, may reflect the existence of a regulatory mechanism which apportions lipid synthesis between the two pathways in response to alterations in the physical properties of the chloroplast membranes.     

Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology

Summary Mutant lines of Arabidopsis thaliana resistant to the artificial auxin 2,4-dichloro phenoxyacetic acid (2,4-D) were isolated by screening for growth of seedlings in the presence of toxic levels of 2,4-D. Genetic analysis of these resistant lines indicated that 2,4-D resistance is due to a recessive mutation at a locus we have designated Axr-1. Mutant seedlings were resistant to approximately 50-fold higher concentrations of 2,4-D than wild-type and were also resistant to 8-fold higher concentrations of indole-3-acetic acid (IAA) than wild-type. Labelling studies with (¹⁴C)2,4-D suggest that resistance was not due to changes in uptake or metabolism of 2,4-D. In addition to auxin resistance the mutants have a distinct morphological phenotype including alterations of the roots, leaves, and flowers. Genetic evidence indicates that both auxin resistance and the morphological changes are due to the same mutation. Because of the pleiotropic morphological effects of these mutations the Axr-1 gene may code for a function involved in auxin action in all tissues of the plant.