The electrochemical H+ gradient in the yeastRhodotorula glutinis

The electrochemical gradient of protons, , was estimated in the obligatory aerobic yeastRhodotorula glutinis in the pH₀ range from 3 to 8.5. The membrane potential, , was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP⁺) for negative above pH₀ 4.5, and thiocyanate (SCN^–) for positive below pH₀ 4.5. The chemical gradient of H⁺ was determined by measuring the chemical shift of intracellular P_i by³¹P-NMR at given pH₀ values. The values of pH_i increased almost linearly from 7.3 at pH₀ 3 to 7.8 at pH₀ 8.5. In the physiological pH₀ range from 3.5 to 6, was fairly constant at values between 17–18 KJ mol^–1, gradually decreasing at pH₀ above 6. In deenergized cells, the intracellular pH_i decreased to values as low as 6, regardless of whether the cell suspension was buffered at pH₀ 4.5 or 7.5. There was no membrane potential detectable in deenergized cells.     

1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator O _R ³ oligonucleotide, which is the preferential binding site for the Cro repressor of phage , was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1 and the H2-H2 sugar protons of the O _R ³ operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the O _R ³ operator — Cro repressor interaction.Abbreviations COSY correlated spectroscopy - FID free induction decay - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect spectroscopy - RD relaxation delay - TSP sodium 3-trimethylsilyl-(2,2,3,3-²H₄)propionate - EDTA sodium ethylendiamine tetraacetate     

In vitro metabolism of N-methyl-dibenzo [c,g]carbazole a potent sarcomatogen devoid of hepatotoxic and hepatocarcinogenic properties

The metabolism of N-methyl substituted 7H-dibenzo[c,g]carbazole (N-Me DBC) was investigated in vitro using liver microsomes from 3-methylcholanthrene (MC)-, benzo[c]carbazole (BC) and Arochlor-pretreated mice and rats. N-Me DBC is a potent sarcomatogen devoid of hepatotoxicity and liver carcinogenic activity. The ethyl acetate-extractable metabolites were separated by high performance liquid chromatography (HPLC) and most of them were identified by proton magnetic resonance (PMR), mass spectrometry (MS) and comparison with synthetically prepared specimens. Mouse and rat microsomes gave rise to the same metabolites. The major metabolites were 5-OH-N-Me DBC (50%), N-hydroxymethyl (HMe) DBC (25-30%) and 3-OH-N-Me DBC (10%). Addition of 1,1,1-trichloropropene-2,3-oxide (TCPO) to the standard incubation medium permitted the identification of two dihydrodiols among the minor metabolites. No metabolite of DBC was observed after incubation of N-Me DBC, or its major metabolite N-HMe DBC, with either mouse or rat microsomes, but the possibility of a slight demethylation cannot be totally excluded. The lack of biotransformation at the nitrogen atom site may explain the lack of hepatotoxicity and liver carcinogenic activity of N-Me DBC. The modulation of metabolism by epoxide hydrolase, cytosol and glutathione was also investigated. The results are discussed in the light of data previously obtained with hepatotoxic and hepatocarcinogenic DBC.     

Aminoacyl-tRNA synthetases: inter-site interaction as a possible proofreading mechanism

Smooth muscle cell energetics of taenia caeci during relaxation, activity and maximal contraction were investigated using ³¹P-NMR. In relaxed muscle obtained in calcium-free medium, [ATP], [phosphocreatine] and [sugar phosphate] were 4.4 mM, 7.7 mM and 2.8 mM, respectively. There was only a small difference in the energetics of spontaneously active and maximally contracted muscles, but under both conditions substantial changes occurred as compared with relaxed muscles. The internal pH in relaxed muscle was found to be 7.05, which acidified to 6.5 during contraction. The level of sugar phosphates was found to be not a limiting factor in energetics.     

The effects of bisulfite on growth and macromolecular synthesis in Escherichia coli

Bisulfite reversibly inhibits the growth of a variety of microorganisms and has been used as a preservative in foods and beverages for that reason. We have now measured macromolecule synthesis in Escherichia coli K12 after bisulfite treatment. RNA synthesis, the synthesis of total protein, and of an inducible enzyme, beta-galactosidase, stopped almost immediately upon addition of 2 mM (or higher concentrations) of bisulfite. These functions resumed after a lag whose duration depended on the concentration of bisulfite added. The synthesis of DNA was slowed upon bisulfite addition, but did not stop entirely. The inhibition of RNA synthesis by bisulfite took place in both stringent and relaxed strains of E. coli and was not relieved upon addition of chloramphenicol. Stringent control was therefore not involved in this effect. No effect on protein synthesis was observed in the cell-free system of E. coli (using poly(U) or MS2 RNA as messenger) at bisulfite concentrations up to 10 mM. Protein synthesis inhibition in vivo was apparently not due to a reaction of bisulfite with a component of this system. In additional experiments, RNA polymerase was not impaired by bisulfite, and the growth inhibition effect was shown to proceed in the presence of inhibitors of free radical chain reactions.     

Effects of Hypoglycaemia and Hypoxia on the Intracellular pH of Cerebral Tissue as Measured by 31P Nuclear Magnetic Resonance

Changes in high-energy phosphate metabolites and the intracellular pH (pHi) were monitored in cerebral tissue during periods of hypoglycaemia and hypoxia using 31P nuclear magnetic resonance spectroscopy. Superfused brain slices were loaded with deoxyglucose at a concentration shown not to impair cerebral metabolism, and the chemical shift of the resulting 2-deoxyglucose-6-phosphate (DOG6P) peak was used to monitor the pHi. In some experiments with low circulating levels of Pi, the intracellular Pi was visible and indicated a pH identical to that of DOG6P, an observation validating its use as an indicator of pHi in cerebral tissue. The pHi was found to be unchanged during moderate hypoglycaemia; however, mild hypoxia (PO2 = 16.4 kPa) and severe hypoglycaemia produced marked reductions from the normal of 7.2 to 6.8 and 7.0, respectively. Hypoglycaemia caused a fall in the level of both phosphocreatine (PCr) and ATP, whereas hypoxia affected PCr alone, as shown previously. However, the fall in pHi was similar during the two insults, thus indicating that the change in pH is not directly linked to lactate production or to the creatine kinase reaction.     

L7811鼠腹水肿瘤细胞^31P核磁共振的研究

用~(31)P核磁共振技术(~(31)P-NMR)研究了L_(7811)鼠腹水肿瘤细胞和615系鼠胸腺细胞(正常对照细胞)。结果发现在肿瘤晚期阶段,L_(7811)腹水肿瘤细胞的含磷化合物未进入完全不活跃状态。此外,腹水肿瘤细胞的磷脂组成与含量亦有明显改变。因此,~(31)P-NMR谱可做为观察肿瘤细胞内能量生成和某些磷脂合成宏观动态过程的一项参考指标。     

Na~(+)、Ca~(2+)、Cu~(2+)和Zn~(2+)与HSA相互作用的~(23)(Na)-NMR研究

 本文应用~23Na-NMR波谱技术,研究了Na~(+)、Ca~(2+)、Cu~(2+)和Zn~(2+)与人体血清白蛋白(HSA)的相互作用。在实验基础上,通过引入两位快交换模型,拟合计算获得了Na~(+)与HSA相互作用的结合常数和处于结合状态Na~(+)的相关时间;实验表明Ca~(2+)能与Na~(+)竞争同HSA结合,拟合计算获得了两者与HSA相互作用结合常数的比值,棕榈酸钠能增强Ca~(2+)同Na~(+)竞争与HSA结合的能力;从实验上未能观察到Cu~(2+)、Zn~(2+)能同Na~(+)竞争与HSA相互作用的证据。     

Intracellular magnetophoresis of amyloplasts and induction of root curvature

High-gradient magnetic fields (HGMFs) were used to induce intracellular magnetophoresis of amyloplasts. The HGMFs were generated by placing a small ferromagnetic wedge into a uniform magnetic field or at the gap edge between two permanent magnets. In the vicinity of the tip of the wedge the dynamic factor of the magnetic field, (H²/2), was about 10⁹ Oe² · cm^–1, which subjected the amyloplasts to a force comparable to that of gravity. When roots of 2-d-old seedlings of flax (Linum usitatissimum L.) were positioned vertically and exposed to an HGMF, curvature away from the wedge was transient and lasted approximately 1 h. Average curvature obtained after placing magnets, wedge and seedlings on a 1-rpm clinostat for 2 h was 33 ± 5 degrees. Roots of horizontally placed control seedlings without rotation curved about 47 ± 4 degrees. The time course of curvature and changes in growth rate were similar for gravicurvature and for root curvature induced by HGMFs. Microscopy showed displacement of amyloplasts in vitro and in vivo. Studies with Arabidopsis thaliana (L.) Heynh. showed that the wild type responded to HGMFs but the starchless mutant TC7 did not. The data indicate that a magnetic force can be used to study the gravisensing and response system of roots.Abbreviations HGMF high-gradient magnetic field - emu electromagnetic units - Oe Oersted We thank Dr. John Kiss, Miami University, Ohio for providing the Arabidopsis seeds. This work was supported by NASA grant NAGW-3656