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.     

Translational regulation of the synthesis of a major heat shock protein in HeLa cells

The synthesis of a major heat shock protein (HSP 70) was measured in HeLa cells incubated at 42.5 degrees C and then transferred to 37 degrees C or 30 degrees C. After 90 min, synthesis of HSP 70 decreased by 54 and 85%, respectively, whereas HSP 70 mRNA was reduced at most by 20%. Therefore, the reduced synthesis of HSP 70 could not be accounted for by mRNA turnover. HSP 70 was associated with large polyribosomes (6-10 ribosomes) in cells kept at 42.5 degrees C, but with medium or small polyribosomes in cells transferred to 37 degrees C or 30 degrees C (5-6 or 2-3 ribosomes, respectively). Addition of puromycin to these cells resulted in the release of all ribosomes from HSP 70 mRNA, indicating that they were translationally active. The regulation of HSP 70 synthesis was investigated in cell-free systems prepared from heat-shocked or control cells and incubated at 30 degrees C and 42 degrees C. After 5 min at 42 degrees C, the cell-free system from heat-shocked cells synthesized protein at 3 times the rate of the control cell-free system. This difference was in large part due to synthesis of HSP 70. Addition of HSP mRNA to the control cell-free system stimulated protein synthesis at 42 degrees C, but not at 30 degrees C. These findings suggest that translation of HSP 70 mRNA is specifically promoted at high temperature and repressed during recovery from heat shock by regulatory mechanisms active at the level of initiation.     

Modifications evoked by piretanide in the mechanical activity of cardiovascular tissues

The effects of piretanide upon mechanical activity and pharmacological reactivity of vascular and myocardial tissues from normotensive rats were investigated. Magnitude of phasic contractions of isolated rat portal vein was diminished by the drug in a dose-related manner; contractile depression induced by piretanide (10(-4)M) was less in the presence of insulin (0.1 U/mL), glucose (22 mM) or pyruvate (5 mM). Responses to KCl (90 mM), or norepinephrine (2.5 X 10(-5)M) were also reduced. Contractile activity of atria and ventricle strips was diminished only when piretanide reached 10(-4)M. Results support direct actions of piretanide upon cardiac and vascular tissues. Possible mechanisms of action are discussed.     

Photoaffinity labelling of androgen receptors with 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preparations.     

Effect of chemical modifications on the K99 and K88ab fibrillar adhesins of Escherichia coli

The role of specific amino acid residues of the K88ab and K99 fibrillar adhesins in the binding to erythrocytes and antibodies has been studied by chemical modification. It appeared that: (1) The integrity of the single disulfide bridge in the K99 subunits is essential for the binding of the fibrillae to the glycolipid receptors, but not for the recognition and binding of specific anti-K99 antibodies. (2) Modification of one lysine residue per subunit with 4-chloro-3,5-dinitrobenzoate results in the loss of the adhesive capacity of K99 fibrillae. Lysine residue are not important for the adhesive activity of K88ab fibrillae. Three or five lysine residues per subunit, respectively, can be modified without an effect on the immunological properties of the K99 and K88ab fibrillae. (3) Limited reaction of K99 and K88ab fibrillae with 2,3-butanedione destroys the adhesive activity of both fibrillae. This inactivation corresponds with the loss of one (K99) or two (K88ab) arginine residues per subunit. Ultimately, in K99 three, and in K88ab four, arginine residues per subunit can be modified without affecting the binding of specific antibodies. (4) Modification of five out of the nine carboxyl groups contained in the K99 subunit suppresses the recognition of specific anti-K99 antibodies, but carboxylates are not important for the adhesive activity of K99 fibrillae. Modification of two additional carboxylates in K99 results in an insoluble product. (5) Tyrosine residues are most probably not present in the adhesive or antigenic sites of K99 fibrillae. Modification of six out of the ten tyrosine residues in the K88ab subunit results in a decrease in adhesive activity but has no effect on the reaction with anti-K88ab antibodies.     

Essential adaptation of the calcium influx assay into liposomes with entrapped arsenazo III for studies on the possible calcium translocating properties of acidic phospholipids

An adapted version of the Ca2+-influx assay of Weissmann et al. (Weissmann, G., Anderson, P., Serhan, C., Samuelson, E. and Goodman, E. (1980) Proc. Natl. Acad. Sci. USA 77, 1506-1510) is presented for studies on the possible ionophoretic properties of acidic phospholipids. This method is based on the use of the metallochromic dye arsenazo III enclosed in liposomal vesicles, to indicate the Ca2+ influx. An essential control is introduced to discriminate between Ca2+-arsenazo III complex formation inside the vesicles, as a consequence of Ca2+ influx, and outside the vesicles, as a consequence of arsenazo III leakage from the vesicles. Furthermore, some minor improvements are added, like the use of large unilamellar vesicles instead of multilamellar vesicles, and the use of dual wavelength spectrophotometry. Using this method, it was found that dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidylglycerol, were impermeable to Ca2+. In this system a selective Ca2+ permeability could be induced by the addition of the fungal Ca2+ ionophore A23187. In contrast, dioleoylphosphatidylcholine vesicles, containing 20 mol% dioleoylphosphatidic acid, incubated in the presence of Ca2+ were permeable to both Ca2+ and arsenazo III.     

2H NMR investigation of the organization and dynamics of polyisoprenols in membranes

The polyisoprenols (PIs) dolichol and undecaprenol function as chemical carriers of glycosyl residues in the membrane-directed synthesis of glycoconjugates in prokaryotic and eukaryotic cells. The molecular details of how these lipid cofactors function is unknown. Presented here are results of deuterium NMR investigations of site specifically 2H-labeled PIs incorporated into model membranes. To complement previous omega-terminal PI labeling schemes, a simple synthesis of head group 2H-labeled PIs is presented in which a PI alcohol is esterified with deuterated acetyl chloride. The 2H-labeled PIs, when incorporated into multilamellar membranes composed of phosphatidylcholine, gave rise to 2H NMR powder patterns interpretable in terms of quadrupole splittings (delta vQ) and spin-lattice relaxation times (T1s). Pure isomers of head group 2H-labeled geraniol (C10) and solanesol (C45) gave rise to single splittings while farnesol (C15) gave rise to two sets of splittings due to cis-trans isomerization at the polar terminal double bond. Membranes containing C45 solanesol exhibited a large isotropic component, indicative of limited partitioning of this poly trans PI into the membrane. T1 measurements revealed high rates of motion for PIs relative to cholesterol in similar membrane hosts and revealed correlation times close to the fatty acyl methyl termini in phosphatidylcholine. The smaller PIs showed higher rates of motion but the T1s of head and tail labels were similar. These data indicate that both ends of the esterified PI molecules see similar environments, probably in the bilayer interior, and suggest that the esterified PIs studied here do not appear to adopt a conventional head group-at-interface orientation of lipids within the bilayer.     

Beneficial effect of S-adenosyl-L-methionine in lead intoxication. Another approach to clinical therapy

Five patients with chronic lead intoxication were treated with S-adenosyl-L-methionine (12 mg/kg body weight, daily), given intravenously, over a period of 22 days. A significant recovery of erythrocytic ALA-D was observed in all cases, after therapy. Blood lead content significantly pathologic at the beginning of SAM administration, rapidly decreased within 24-48 h of initiating treatment, reaching nearly control values at the end of the trial. A good correlation between recovery of ALA-D activity and decreased concentration of lead in RBC was found. GSH content in blood was diminished in lead poisoned patients, increasing to normal levels after SAM administration. Other biochemical parameters such as Deaminase activity in RBC, ALA, PBG, porphyrins and lead in urine and serum gamma-GT were measured, showing no important deviations from control values before, during or after treatment. Both biochemical and clinical improvement was observed, indicating that SAM therapy is beneficial in the treatment of lead intoxication. No untoward signs were observed. The mechanism of action of SAM is not yet clear; however, a chelating effect could be excluded, and very likely its action can be attributed to glutathione availability.