Cloning and characterization of a linear 2.3 kb mitochondrial plasmid of maize

Summary A linear 2.3 kb DNA molecule found in maize mitochondria was cloned into pUC8. A natural deletion of this plasmid, found in cmsT and some N (fertile) types of maize plants, was mapped to one end of the plasmid. A minor sequence homology to S-2, another linear mitochondrial plasmid, was detected, as well as more significant sequence homology with chloroplast and maize nuclear DNA. Hybridization to teosinte mitochondrial DNA (mtDNA) revealed the presence of part of the maize plasmid in the high molecular weight mtDNA of the maize relatives. RNA dot hybridization indicates that the plasmid is transcribed in mitochondria. The termini of the 2.3 kb linear plasmid contain inverted repeated sequences; of the first 17 nucleotides of the termini, 16 are identical to the terminal inverted repeats of the linear S plasmids found in the mitochondria of cmsS maize plants.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

Decreased Muscarinic Acetylcholine Receptor Number in the Central Nervous System of the Tottering (tg/tg) Mouse

The tottering mouse (tg/tg) is a single-locus mutant, phenotypically characterized by the development of epilepsy associated with distinct electroencephalographic abnormalities. Because of reported alterations in muscarinic receptor (mAChR) number in various seizure states, mAChR density was examined in discrete brain regions of tottering (tg/tg) and coisogenic wild-type (+/+) mice. Saturation binding experiments revealed a widespread decrease in membrane mAChR density in the CNS of adult tottering (tg/tg) mice as compared with age-matched control wild-type (+/+) mice. The decrease was most pronounced in the hippocampus, where tg/tg mice exhibited a 40-60% reduction in mAChR density with no change in the affinity of the receptor for antagonists or agonists. At postnatal day 10, before the reported onset of electroencephalographic abnormalities, 114 and 65% increases in mAChR density were observed in the tg/tg hippocampus and cortex, respectively. Following the development of seizure activity at postnatal day 22, mAChR density in the tg/tg hippocampus was reduced by 29%. No change in brain mAChR density was seen in adult heterozygotes (+/tg), which do not develop electroencephalographic or seizure abnormalities. These results indicate that the development of reduced mAChR number in the CNS of the tg/tg mouse is secondary to abnormal neuronal activity, providing further support for the hypothesis that membrane depolarization can cause a decrease in neuronal mAChR density.     

Biofeedback,voluntary control,and human potential

This paper examines some of the philosophical and scientific relationships involving self-control, voluntary control, and psychophysiologic self-regulation. The role of biofeedback in mediating conscious and unconscious processes is explored. Demonstrations of superior voluntary control and its relationship to belief, confidence, and expectation are examined. Biofeedback demonstrates the potential of control to oneself, creating confidence in one's ability to establish enhanced and peak performance in athletics, education, and psychophysiologic therapy. Emphasis is placed on the power of images in all human functioning, and in enhancing human potential.Presidential address presented at the meetings of the Biofeedback Society of America, March 23, 1986, San Francisco.     

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gk^a and Gk^b, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent K _m values for MgATP^2– and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.This work has been supported in part by a grant from the British Diabetic Association and a Training Studentship to PAJ from the Medical Research Council (U.K.).     

Adaptation to High-Intensity, Low-Wavelength Light among Surface Blooms of the Cyanobacterium Microcystis aeruginosa

Natural populations of the nuisance bloom cyanobacterium Microcystis aeruginosa obtained from the eutrophic Neuse River, N.C., revealed optimal chlorophyll a-normalized photosynthetic rates and resistance to photoinhibition at surface photosynthetically active radiation (PAR) intensities. At saturating PAR levels these populations exhibited higher photosynthetic rates in quartz than in Pyrex vessels. Eucaryotic algal populations obtained from the same river failed to counteract photoinhibition. At saturating PAR levels, such populations generally yielded lower photosynthetic rates in quartz containers than they did in Pyrex containers. Cultivation of natural Microcystis populations under laboratory conditions led to physiologically distinct populations which had photoinhibitory characteristics similar to those of other cultured cyanobacterial and eucaryotic algae. Our findings indicate that (i) photosynthetic production among natural surface populations is best characterized and quantified in quartz rather than Pyrex incubation vessels; (ii) extrapolation of natural photoinhibitory trends from laboratory populations is highly subjective to culture and PAR histories and may yield contradictory results; and (iii) buoyant surface-dwelling populations, rather than exhibiting senescence, are poised at optimizing PAR utilization, thereby maintaining numerical dominance in eutrophic waters when physico-chemical conditions favor bloom formation.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

Formation of a 55 000-weight cross-linked beta crystallin dimer in the Ca2+-treated lens. A model for cataract

Incubation of lens in Ca2+-containing media, considered by several investigators to be a useful model of cataract formation, gave rise to significant alterations in the covalent structures of various proteins. In rabbit lens, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used after reduction of disulfides in urea, the most readily observable changes were (i) disappearance of 210K, 95K, and 60K proteins, (ii) modifications of alpha crystallin subunits, (iii) alterations of beta H crystallins, and (iv) de novo production of 55K and higher molecular weight polymers. The addition of leupeptin inhibited the disappearances of 210K, 95K, and 60K proteins and the alteration of alpha crystallins, suggesting that all these were caused by a Ca2+-activated protease. The proteolytically sensitive 60K species was identified as vimentin, a component of intermediate filaments. Formation of the 55K material and of higher molecular weight polymers during Ca2+ treatment of the lens could be prevented by histamine, a compound known to inhibit the transglutaminase-mediated cross-linking of proteins by epsilon-(gamma-glutamyl)lysine peptide bonds in other biological systems. It could also be shown by immunoblotting that an antibody raised against the 55K material reacted selectively with beta crystallins of normal lens. This indicates that the 55K product is in all likelihood an essential intermediate toward higher polymers and that the 55K product is a cross-linked dimer of certain polypeptides of beta crystallin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine specificity, structure, and proteolytic susceptibility of the human lymphocyte receptor for IgE

Properties of the Fc receptor for IgE (FC epsilon R) on cultured human B lymphoblastoid cells (RPMI 8866) were studied. Specificity for human IgE (hIgE) was demonstrated by inhibition studies with both Fc epsilon R+ intact cell and detergent-solubilized receptor preparations. No interaction of the FC epsilon R with other hIg classes or with rodent IgE was seen. In other studies, 3,3-dithiobis(sulfosuccinimidyl) propionate was used to cross-link hIgE to 125I surface-labeled 8866 cells. After detergent solubilization, the 125I receptor components were isolated by immunoprecipitation, and receptor peptides of 83 and 46 kilodalton kD were demonstrated by SDS-PAGE in the presence of reducing agents. Cross-linking performed after detergent solubilization gave identical results. Tryptic maps of the 83 and 46 kD polypeptides were identical with respect to surface-iodinated peptides; this indicates a structural homology between these components. The 83 kD component was more difficult to elute from IgE affinity columns, potentially because of an increased number of IgE binding sites per FC epsilon R molecule. Limited proteolysis studies of the purified FC epsilon R with papain and V8 protease from Staphylococcus aureus demonstrated that a 16 kD FC epsilon R fragment was rapidly produced. This component was also seen after papain treatment of intact cells, and it retained the ability to interact with anti-FC epsilon R antisera and, at least in the absence of detergent, with hIgE affinity columns. Potential relationships between the FC epsilon R and lymphokines that modulate the IgE response (IgE-binding factors) are discussed.