Carbon-13 NMR relaxation studies of pre-melt structural dynamics in [4-13C-uracil] labeled E. coli transfer RNAIVal.

We report 67.8 MHz carbon-13 spin-lattice relaxation studies on [4-13C-uracil] labeled tRNAIVal purified from E. coli SO-187. Following 13C-enriched C4 carbonyl resonances from modified and unsubstituted uridines scattered throughout the polymer backbone enables us to determine dynamical features in both loop and helical stem regions. The experimental results have been analyzed in terms of a model of isotropic overall molecular reorientation. "Anomalous" residues for which the experimental data cannot be accounted for in terms of the model provide an assessment of local and regional properties. Thus, "native" tRNAIVal under physiological conditions of magnesium (10 mM) and temperature (20 degrees - 40 degrees C), exhibits the following characteristics: 1) uridines held rigidly in helical stems and tertiary interactions display correlation times for rotational reorientation of 15-20 nsecs, typical for overall tRNA motion; 2) uridines in loops such as the wobble residue uridine-5-oxyacetic acid (V34) are quite accessible to solvent; moreover V34 and another loop residue, D17, exhibit local mobility; 3) the tertiary interactions involving 4-thio uridine (s4U8) and A14 and ribothymidine (rT54) and A58 are weakened as temperature increases.     

Light-dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5′-triphosphate

Addition of ATP to chloroplasts causes a reversible 25–30% decrease in chlorophyll fluorescence. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU and electron acceptors and has a half-time of 3 minutes. Electron donors to Photosystem I can not overcome the inhibitory effect of DCMU, suggesting that light activation depends on the reduced state of plastoquinone. Fluorescence emission spectra recorded at ?196°C indicate that ATP treatment increases the amount of excitation energy transferred to Photosystem I. Examination of fluorescence induction curves indicate that ATP treatment decreases both the initial (F_o) and variable (F_v) fluorescence such that the ratio of F_v to the maximum (F_m) yield is unchanged. The initial sigmoidal phase of induction is slowed down by ATP treatment and is quenched 3-fold more than the exponential slow phase, the rate of which is unchanged. A plot of F_v against area above the induction curve was identical plus or minus ATP. Thus ATP treatment can alter quantal distribution between Photosystems II and I without altering Photosystem II-Photosystem II interaction. The effect of ATP strongly resembles in its properties the phosphorylation of the light-harvesting complex by a light activated, ATP-dependent protein kinase found in chloroplast membranes and could be the basis of physiological mechanisms which contribute to slow fluorescence quenching in vivo and regulate excitation energy distribution between Photosystem I and II. It is suggested that the sensor for this regulation is the redox state of plastoquinone.     

Cerebral Glutamic Acid Decarboxylase Activity and γ-Aminobutyric Acid Concentrations in Mice Susceptible or Resistant to Audiogenic Seizures

Abstract: DBA/2 mice between 21 and 28 days of age are highly susceptible to sound-induced seizures. Drug studies suggest a possible deficit of γ-Aminobutyric acid (GABA)-mediated neurotransmission may be involved. We have measured the whole brain GABA concentration and glutamic acid decar-boxylase activity in DBA/2 mice at various ages before, during, and after the period of maximal susceptibility to audiogenic seizures. Corresponding determinations were carried out on age-matched TO mice, a strain much less susceptible to audiogenic seizures than DBA/2 mice at all ages. No significant differences in GABA concentration or glutamic acid decarboxylase activity were found between strains at any age. The susceptibility of DBA/2 mice to audiogenic seizures does not result from a gross inability to synthesise or store GABA.     

Enzymic lipid peroxidation in the microsomal fraction of rat brain

Abstract: An enzymic lipid peroxidation system has been demonstrated in the microsomal fraction of rat brain and the requirements and optimal conditions for assay determined. The involvement of NADPH-cytochrome c reductase was demonstrated in vesicles reconstituted with lipids extracted from the brain microsomal fraction. Further characterization of the system made use of substances shown to inhibit the liver microsomal system. α-Tocopherol was shown to be an effective inhibitor of lipid peroxidation in the brain microsomal system, whereas Na₂SO₃ had no effect, which is indicative that free radical transfer occurs only in the hydrophobic regions. Neither superoxide dismutase nor catalase inhibited lipid peroxidation. The implications of an NADPH-cytochrome c reductase-dependent lipid peroxidation system that is not linked to a drug hydroxylation system and appears to differ from the liver microsomal system in a number of other ways are discussed.     

Evidence for a dual action of converting enzyme inhibitor on blood pressure in normal man

We studied the effect of a converting enzyme inhibitor (CEI), Captopril SQ 14,225 50 mg p.o. in eight supine normal subjects under a high sodium (150 mEq/d) and low sodium (25 mEq/d) diet. On high sodium, plasma renin (PRA) and aldosterone were basal and Saralasin did not lower mean blood pressure. However, CEI induced an 11.4 +/- 3.2 mm fall in blood pressure (p less than 0.02) and either indomethacin 50 mg or ibuprofen 800 mg (PI), when given simultaneously on another day abolished the blood pressure response (2.5 +/- 0.9 mm Hg, p greater than 0.5). In contrast, on a low salt diet where renin was increased, CEI induced a drop in blood pressure which was not significantly altered by PI (12.8 +/- 1.1 vs. 10.0 +/- 3.1 mm Hg, p greater than 0.5). CEI increased plasma renin on both diets (1.7 +/- 0.5 to 3.5 +/- 0.8 and 2.8 +/- 0.6 to 12.5 +/- 3.1 ng/ml/hr respectively both p less than 0.05). Aldosterone did not change (high Na+) or fell (low Na+). Inhibition of Prostaglandin synthesis did not significantly block the renin rise from CEI suggesting that the direct angiotensin II negative feedback is relatively independent of acute prostaglandin release. Our studies suggest that CEI has a dual hypotensive action. In a low renin state, the hypotensive action appears to be mediated through vascular prostaglandins.     

Carbon-13 NMR studies on [4-13C] uracil labelled E. coli transfer RNA1(Val1).

In this paper we describe carbon-13 nuclear magnetic resonance results on 13C-enriched purified transfer RNAI(VAL) from from E. coli SO-187, a uracil requiring auxotroph. The organism was grown on uracil 90% 13C-enriched at the carbonyl C4 position. Transfer RNAI(Val) was purified from bulk tRNA by sequential chromatography on columns of BD cellulose, DEAE-Sephadex A-50 and reverse gradient sepharose 4B. Dihydrouridine, 4-thiouridine, and uridine 5-oxyacetic acid located at discrete positions in the polymer backbone were tentatively assigned in the highly resolved 25 MHz 13C-spectra. Chemical shift versus temperature plots reveal differential thermal perturbation of the ordered solution structure, evident in the large dispersion (ca 3-4 ppm) of the uridine C4 resonances. Over the range 26-68 degrees C, V in the anticodon displays the largest downfield shift. Whereas several uridine residues rapidly shift downfield between 50-68 degrees, one moves upfield beginning at 37 degrees. The results are qualitatively compared with proton NMR analysis of the three dimensional structure.     

An Embedding Method for Histochemical Studies of Undecalcified Skeletal Growth Plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tissues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

P nuclear magnetic resonance spectroscopy of lipopolysaccharides from pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by ³¹P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the ‘core’ oligosaccharide region. The spectral signals for various ortho- and pyro-phosphoric esters were observed. All phosphate groups appeared to be mono-esterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.     

31P nuclear magnetic resonance spectroscopy of lipopolysaccharides from pseudomonas aeruginosa

Intact lipopolysaccharide antigens isolated from seven different immunotypes of Pseudomonas aeruginosa have been examined by ³¹P-NMR spectroscopy. These macromolecular complexes contain phosphorus covalently attached to the carbohydrate residues present in the lipid A moiety and the ‘core’ oligosaccharide region. The spectral signals for various ortho- and pyro-phosphoric esters were observed. All phosphate groups appeared to be mono-esterified. Certain shifts characteristic for phosphate diester groups, observed in lipopolysaccharide complexes from other Gram-negative bacteria, were absent. Furthermore, no evidence was found to indicate that phosphate groups are involved in the covalent linkage of individual lipopolysaccharide complexes to form dimers or trimers.