Neocarzinostatin chromophore: Presence of a cyclic carbonate subunit and its modification in the structure of other biologically active forms

On the basis of spectroscopic evidence, opening of a five-membered cyclic carbonate ring (1,3-dioxolan-2-one) in the C₁₅-subunit of the previously determined partial structure $\text{1}$ (Fig. 1) of the major neocarzinostatin chromophore (NCS-Chrom $\text{A}$), is proposed to account for its base-catalyzed methanolysis to NCS-Chrom $\text{C}$. NCS-Chrom $\text{B}$, apparently an authentic natural product present as a minor component in all preparations of NCS studied, was found to be formally equivalent to the hydrolysis/decarboxylation product of the cyclic carbonate functionality in NCS-Chrom $\text{A}$. The mercaptan-dependent DNA strand-scission activity, equivalent for NCS-Chrom $\text{A}$, $\text{B}$ and $\text{C}$, is independent of the integrity of the cyclic carbonate ring system and implicates a secondary site in the C₁₅-substructure for mercaptan activation.     

Carbon-13 nuclear magnetic resonance studies of acetate metabolism in intact cells of Rhodopseudomonas sphaeroides

¹³C-nuclear magnetic resonance was used to study the metabolism of [2-¹³C]acetate in suspensions of Rhodopseudomonas sphaeroides. In the dark, in logarithmic-phase cells the ¹³C label appeared first in butyrate C-2 and C-4 and subsequently in glutamate C-4 and succinate C-2 and C-3. In the light, synthesis of poly(β-hydroxybutyrate) (PHB) takes place. Butyrate synthesis seems to be independent of PHB synthesis or degradation activity. Starved, logarithmic-phase cells also show massive synthesis of PHB in the dark. Stationary-phase cells incorporate ¹³C predominantly into glutamate and succinate. No significant butyrate biosynthesis can be detected in the dark or during illumination. The incorporation of label in PHB is very slow in these cells and most probably originates from exchange of ¹²C for ¹³C into PHB. This might indicate slow turnover without net synthesis of the polymer occurring under these conditions. The results are discussed in relation to the redox state and the availability of metabolic energy for biosynthetic reactions in the dark and during illumination of cell suspensions of Rps. sphaeroides.     

Quantitative assessment of neurochemical changes in a rat model of long-term alcohol consumption as detected by in vivo and ex vivo proton nuclear magnetic resonance spectroscopy

The aim of present study was to quantitatively investigate the neurochemical profile of the frontal cortex region in a rat model of long-term alcohol consumption, by using in vivo proton magnetic resonance spectroscopy (¹H-MRS) at 4.7 T and ex vivo¹H high-resolution magic angle spinning (HR-MAS) technique at 11.7 T. Twenty male rats were divided into two groups and fed a liquid diet for 10 weeks. After 10 weeks, in vivo¹H MRS spectra were acquired from the frontal cortex brain region. After in vivo¹H MRS experiments, all animals were sacrificed and 20 frontal cortex tissue samples were harvested. All tissue examinations were performed with the 11.7 T HR-MAS spectrometer and high-resolution spectra were acquired. The in vivo and ex vivo spectra were quantified as absolute metabolite concentrations and normalized ratios of total signal-intensity (i.e., metabolites_Norm), respectively. The absolute quantifications of in vivo spectra showed significantly higher glycerophosphocholine plus phosphocholine (GPC + PCh) and lower myo-inositol (mIns) concentrations in ethanol-treated rats compared to controls. The quantifications of ex vivo spectra showed significantly higher PCh_Norm, Cho_Norm and tCho_Norm, and lower GPC_Norm and mIns_Norm ratio levels in ethanol-treated rats compared to controls. Our findings suggest that reduced mIns concentrations caused by the long-term alcohol consumption may lead to hypo-osmolarity syndrome and astrocyte hyponatremia. In addition, increased choline-containing compound concentrations may reflect an increased cell turnover rate of phosphatidylcholine and other phospholipids, indicating an adaptive mechanism. Therefore, these results might be utilized as key markers in chronic alcohol intoxication metabolism.     

Distribution and radical scavenging activity of phenols in Ascophyllum nodosum (Phaeophyceae)

Phlorotannins have been purified and fractionated in the brown alga Ascophyllum nodosum using successively differential extraction, liquid-liquid separation and dialysis. Both the phenol content and the radical scavenging capacity of the resulting fractions were assayed by the Folin-Ciocalteu test and the DPPH method, respectively, whilst purity of the fractions was assessed by ¹H NMR analysis. The purification process resulted in the isolation of six fractions from each crude extract with only minor losses. High levels of phenols, up to 97-99%, were measured in semi-purified fractions containing phlorotannins more than 50 kDa in average molecular size, accounting for more than 95% of the ethyl acetate phenol pool. As a consequence, purity decreased in ethyl acetate fractions together with the molecular size of compounds. The importance of differential extraction based on the polarity of phenols is highlighted by the fact that most of these compounds were found in the ethyl acetate fraction after the first extraction step in 100% methanol, whilst two thirds of phenols extracted by 50% methanol remained in the aqueous phase. The radical scavenging activity of the fractions was correlated with the phenol content and was maximal in complete ethyl acetate fractions and in dialysis concentrates containing molecules more than 50 kDa in size. The specific activity of phenols was found to be maximal for molecules smaller than 2 kDa when isolated from the 100% methanol extract and 1-4 times smaller in the water phase separated from the same extract. The distribution of radical-scavenging potentials in the phenol pool of A. nodosum supports the idea that physiological roles and putative uses of phlorotannins are under the control of a polarity-molecular size complex.     

NMR structure of an intracellular third loop peptide of human GABA(B) receptor

GABA_B receptor is a G protein-coupled receptor for GABA and drug target for neurological and psychiatric disorders. From the analysis of GTPγS binding assay, we found that a synthesized peptide (GABAb: ETKSVSTEKINDHR) corresponding to the intracellular third loop region of metabotropic GABA_B receptor could activate G_i protein α subunit directly. The three dimensional molecular structure of the peptide in SDS-d₂₅ micelles was determined by 2D ¹H-NMR spectroscopy. GABAb peptide formed an α helical structure and a positive charge cluster at the C-terminal site. These structural features were also found in several other G protein activating peptides. From the comparison among these peptides, we found that peptides with high helical content show the high activity.     

Induction of mitotic gene conversion in Saccharomyces cerevisiae by N-nitrosated pesticides

The herbicide N-(2-benzothiazolyl)-N′-methylurea (Benzthiazuron, Gatnon®) the insecticides 2-isopropoxyphenyl-N-methylcarbamate (Propoxur, Unden®) and 1-naphthyl-N-methylcarbamate (Carbaryl, Sevin®), together with their N-nitroso derivatives, were examined for genetic activity. A diploid strain of Saccharomyces cerevisiae heteroallelic at the gene loci ade2 and trp5, was used to test for the induction of mitotic gene conversion in these two unlinked gene loci. The non-nitrosated compounds had no influence on the frequency of mitotic gene conversions. The nitrosated substances, however, displayed marked convertogenic activities. N-nitrosopropoxur (N-nitrosopropoxur, N-nitrosocarbaryl) were much more active than the substituted N nitrosomethylurea (N-nitrosobenzthiazuron). N-Nitrosocarbaryl induced the greatest number of mitotic gene conversions.     

pH dependence of amide chemical shifts in natively disordered polypeptides detects medium-range interactions with ionizable residues

A growing number of natively disordered proteins undergo a folding/binding process that is essential for their biological function. An interesting question is whether these proteins have incompletely solvated regions that drive the folding/binding process. Although the presence of predominantly hydrophobic buried regions can be easily ascertained by high-sensitivity differential scanning calorimetry analysis, the identification of those residues implicated in the burial requires NMR analysis. We have selected a partially solvated natively disordered fragment of Escherichia coli, thioredoxin, C37 (38-108), for full NMR spectral assignment. The secondary chemical shifts, temperature coefficients, and relaxation rates (R(1) and R(2)) of this fragment indicate the presence of a flexible backbone without a stable hydrogen bond network near neutral pH. (1)H-(15)N heteronuclear single quantum coherence analysis of the pH dependence of amide chemical shifts in fragment C37 within pH 2.0 and 7.0 suggests the presence of interactions between nonionizable residues and the carboxylate groups of four Asp and four Glu residues. The pH midpoints (pH(m)) of the amides in the ionizable residues (Asp or Glu) and, consequently, the shifts in the pH(m) (DeltapH(m)) of these residues with respect to model tetrapeptides, are sequence-dependent; and the nonionizable residues that show pH dependence cluster around the ionizable ones. The same pH dependence has been observed in two fragments: M37 (38-73) and C73 (74-108), ruling out the participation of long-range interactions. Our studies indicate the presence of a 15-residue pH-dependent segment with the highest density of ionizable sites in the disordered ensembles of fragments C37 and M37. The observed correlations between ionizable and nonionizable residues in this segment suggest the organization of the backbone and side chains through local and medium-range interactions up to nine residues apart, in contrast to only a few interactions in fragment C73. These results agree qualitatively with the predominantly hydrophobic buried surface detected only in fragments C37 and M37 by highly sensitive differential scanning calorimetry analysis. This work offers a sensitive and rapid new tool to obtain clues about local and nonlocal interactions between ionizable and nonionizable residues in the growing family of natively disordered small proteins with full NMR assignments.     

Raman spectroscopy of liver alcohol dehydrogenase

We report the Raman spectrum of liver alcohol dehydrogenase in solution. The enzyme's secondary structure as determined from an examination of the Raman bands is slightly different than that found in crystals by X-ray diffraction.