Effects of the thyroid status on the sarcoplasmic reticulum in slow skeletal muscle of the rat

The effects of the thyroid status on the Ca++-transporting capabilities of rat slow skeletal muscle (m.soleus) were studied. The oxalate supported Ca++-uptake activity and Ca++-loading capacity of muscle homogenates from hyperthyroid rats showed an approximate 4.2 and 2.5 fold increase, respectively, as compared to values found in the hypothyroid group. Muscle homogenates of euthyroid rats gave intermediate values. The specific activity of oxalate supported Ca++ uptake, but not the Ca++-loading capacity, of membrane preparations enriched with respect to sarcoplasmic reticulum (SR) increased in proportion to the thyroid status. This was paralleled by a 3.5 fold increase in the amount of active Ca++ pumps in the SR preparations in the transition from hypothyroidism to hyperthyroidism as determined by measurement of Ca++-dependent 32P incorporation. These observations are not explained by differences in degree of purification of the examined SR preparations. Protein profiles of the membrane preparations obtained by gel electrophoresis indicated a thyroid-hormone dependent increase in Ca++-pump content relative to other SR proteins. The results suggest that thyroid hormone stimulates the proliferation of the SR and possibly also increases the Ca++-pump density in the SR membrane.     

Synthesis of novel acetylene-containing amino acids

Summary.  Novel synthetic procedures for the modification of non-proteinogenic acetylene-containing amino acids have been developed. The functionalization either proceeds via zinc/copper-mediated introduction of alkyl substituents, or via tungsten-catalyzed ring-closing alkyne metathesis reactions. Received March 28, 2002 Accepted October 3, 2002 Published online December 18, 2002 Acknowledgements These investigations are supported (in part) by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW). Authors' address: Floris P. J. T. Rutjes, Prof. Dr., Department of Organic Chemistry, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands, E-mail: rutjes@sci.kun.nl  2, selected data: ¹H NMR (300 MHz, CDCl₃) δ 5.32 (d, J = 7.7 Hz, 1H), 4.44–4.40 (m, 1H), 3.76 (s, 3H), 2.75–2.73 (d, J = 5.0 Hz, 2H), 1.44 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.0, 155.0, 80.3, 74.6, 52.6, 51.9, 41.7, 28.3, 24.0; mp = 55°C.  Typical procedure for 5: zinc dust (116 mg, 1.408 mmol) was weighed into a 20 mL flask, which was repeatedly evacuated (with heating using a heat gun) and flushed with argon. Dry DMF (0.5 mL, distilled from CaH₂) and 1,2-dibromoethane (9.2 μL, 0.106 mmol) were added and the flask was heated at 80°C for 40 min. The reaction mixture was allowed to cool to room temperature, trimethylsilyl chloride (4 μL, 0.035 mmol) was added and the resulting mixture was stirred vigorously for a further 30 min under argon. Iodocyclohexane (69 μl, 0.528 mmol) was added and stirred at room temperature for 3 h more after which stirring was ceased to settle the zinc. CuCN (41 mg, 0.458 mmol) and LiCl (40 mg, 0.915 mmol) were heated to 150°C for 2 h and cooled to room temperature. Addition of DMF (1 mL) formed a soluble CuCN·2LiCl complex within 5 min. After cooling the Cu-complex to −15°C, the organozinc reagent was added dropwise followed by the bromoacetylene 2 (116 mg, 0.352 mmol). The mixture was allowed to stir overnight at room temperature. Water was added and the suspension was extracted using heptane, washed with brine, dried (MgSO₄) and concentrated. Purification using flash column chromatography (10% EtOAc in heptane) yielded 5 (100 mg, 81%) as a colorless oil. 5: IR ν 3355, 2929, 2852, 2359, 2337, 1749, 1717, 1498, 1447, 1365, 1251, 1181, 1060; ¹H NMR (300 MHz, CDCl₃) δ 5.28 (d, J = 7.7 Hz, 1H), 4.43–4.38 (m, 1H), 3.73 (s, 3H), 2.69–2.63 (m, 2H), 2.13 (m, 1H), 1.73–1.22 (m, 10H), 1.43 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.4, 155.0, 88.1, 79.9, 73.8, 52.3, 32.7, 32.7, 28.8, 28.2, 25.8, 24.6, 23.1; HRMS (EI): calculated for C₁₇H₂₇NO₄ 309.1940, found 309.1937.  A solution of the tungsten catalyst (7 mg, 10 mol%) in C₆H₅Cl (2 mL) was treated with a solution of 14 (49.0 mg, 0.120 mmol) in C₆H₅Cl (5.0 mL) under an argon atmosphere and the resulting mixture was heated at 80°C for 3 h. Evaporation followed by flash column chromatography (80% EtOAc in heptane) afforded 15 (21.0 mg, 50%; 64% after correction for starting material) and 14 (16 mg, 33%) as colorless oils. 15: [α]_D =–14.6 (c = 1, CH₂Cl₂); IR ν 3313, 2931, 2865, 2249, 1744, 1667, 1520, 1366, 1170; ¹H NMR (400 MHz, CDCl₃) δ 7.14 (d, J = 8.7 Hz, 1H), 6.08 (d, J = 8.3 Hz, 1H), 4.78 (q, J = 6.8 Hz, 1H), 4.27 (q, J = 7.9 Hz, 1H), 3.73 (s, 3H), 2.17–2.15 (m, 4H), 2.07–1.96 (m, 2H), 1.79–1.52 (m, 4H), 1.45 (s, 9H), 0.89–0.83 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.2, 171.8, 155.8, 80.4, 80.2, 79.3, 53.8, 52.5, 51.2, 32.8 (2×), 28.1, 24.6, 24.2, 18.3 (2×); HRMS (EI): calculated for C₁₈H₂₈N₂O₅     

Exon skipping in the sterol 27-hydroxylase gene leads to cerebrotendinous xanthomatosis

We report a new mutation in the sterol 27-hydroxylase (CYP 27) gene in a Dutch family with cerebrotendinous xanthomatosis: a G→A transition in the splice donor site in intron 4. This mutation leads to skipping of exon 4, resulting in a loss of 66 amino acids in the CYP 27 enzyme molecule. Received: 15 March 1997 / Accepted: 26 March 1997     

Antibiotic activity of isoxazolylnaphthoquinone imines on mice infected with Staphylococcus aureus

I. ALBESA, P. BOGDANOV, A. ERASO, N.R. SPERANDEO AND M.M. DE BERTORELLO. 1995. The antibiotic activity of new synthetic isoxazolylnaphthoquinone imines was studied. Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were resistant to the four compounds studied (MIC > 128 µg ml⁻¹), but Staphylococcus aureus ATCC 25923, ATCC 29213 and 30 clinical isolates of Staph. aureus were inhibited by 2-hydroxy- N -(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (I). This compound diminished bloodstream infection of mice injected i.m. with Staph. aureus; septicaemia decayed significantly when I was applied at the beginning of the infection while when I was given 3 d after bacterial challenge, a significant protection was afforded. Bactericidal activity in serum increased during the 5 h after I was administered i.p.
The acetyl derivative of I had a high MIC but when inoculated orally in mice decreased the Staph. aureus counts in circulation. This protection occurred only when the schedule of administration started close to the bacterial challenge. Antibiotic activity in vivo may be associated with in vitro effects.     

Conformational analysis of the 3'-5'-cyclic dinucleotide d less than pApA greater than by means of molecular mechanics

The conformational properties of the cyclic dinucleotide d less than pApA greater than were studied by means of molecular mechanics calculations in which a multiconformation analysis was combined with minimum energy calculations. In this approach models of possible conformers are built by varying the torsion angles of the molecule systematically. These models are then subjected to energy minimization; in the present investigation use was made of the AMBER Force field. It followed that the lowest energy conformer has a pseudo-two-fold axis of symmetry. In this conformer the deoxyribose sugars adopt a N-type conformation. The conformation of the sugar-phosphate backbone is determined by the following torsion angles: alpha +, beta t, gamma +, epsilon t and zeta +. The conformation of this ringsystem corresponds to the structure derived earlier by means of NMR spectroscopy and X-ray diffraction. The observation of a preference for N-type sugar conformations in this molecule can be explained by the steric hindrance induced between opposite H3' atoms when one sugar is switched from N- to S-type puckers. The sugars can in principle switch from N- to S-type conformations, but this requires at least the transition of gamma + to gamma -. In this process the molecule obtains an extended shape in which the bases switch from a pseudo-axial to a pseudo-equatorial position. The calculations demonstrate that, apart from the results obtained for the lowest energy conformation, the 180 degrees change in the propagation direction of the phosphate backbone can be achieved by several different combinations of the backbone torsion angles. It appeared that in the low energy conformers five higher order correlations are found. The combination of torsion angles which are involved in changes in the propagation direction of the sugar-phosphate backbone in DNA-hairpin loops and in tRNA, are found in the dataset obtained for cyclic d less than pApA greater than. It turns out, that in the available examples, 180 degrees changes in the backbone direction are localized between two adjacent nucleotides.     

The receptor for transferrin on murine myeloma cells: one-step purification based on its physiology, and partial amino acid sequence

The receptor for transferrin is one of the major surface proteins of proliferating lymphocytes and other cells. It binds ferrotransferrin from serum and endocytoses it into an acidic nonlysosomal intracellular compartment where iron is released, but in which apotransferrin remains tightly bound to its receptor. Recycling of the apotransferrin-receptor complex to the cell surface is associated with a return to neutral pH and concomitant loss of affinity of apotransferrin for its receptor. Apotransferrin is then free to leave the cell and initiate a new cycle. We have exploited this cycle in a novel method for the purification of the receptor for transferrin. Murine myeloma cells were lysed in nonionic detergent, and the lysate passed over a column of ferrotransferrin-agarose at pH 7.4. After washing with sodium acetate at pH 5.0, iron was removed with sodium citrate pH 5.0 and desferrioxamine. Upon returning the pH to neutrality, the receptor was eluted and found to be homogeneous by SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. The degree of purification was estimated to be at least 3,000-fold, and the calculated yield was 10 to 20%. The purified receptor was capable of binding to transferrin. The receptor was digested with trypsin, and the resulting peptides were separated by reversed-phase high performance liquid chromatography in NH4HCO3. Selected peptides were rechromatographed in 0.1% trifluoroacetic acid, and their amino acid sequences were determined.