Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine–guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

Inactivation of Rabbit Liver Carbonyl Reductase by Phenylglyoxal and 2,3,4-Trinitrobenzenesulfonate Sodium

The chemical modifications of rabbit liver carbonyl reductase (RLCR) with phenylglyoxal (PGO) and 2,3,4-trinitrobenzenesulfonate sodium (TNBS), which are respective chemical modifiers of arginine and lysine residues, were examined. RLCR was rapidly inactivated by these modifiers. Kinetic data for the inactivation demonstrated that each one of arginine and lysine residues is essential for catalytic activity of the enzyme. Furthermore, based on the protective effects of NADP +, NAD + and their constituents against the inactivation of RLCR by PGO and TNBS, we propose the possibility that the functional arginine and lysine residues are located in the coenzyme-binding domain of RLCR and interact with the 2′-phosphate group of NADPH.     

What Happened before Losses of Photosynthesis in Cryptophyte Algae?

In many lineages of algae and land plants, photosynthesis was lost multiple times independently. Comparative analyses of photosynthetic and secondary nonphotosynthetic relatives have revealed the essential functions of plastids, beyond photosynthesis. However, evolutionary triggers and processes that drive the loss of photosynthesis remain unknown. Cryptophytes are microalgae with complex plastids derived from a red alga. They include several secondary nonphotosynthetic species with closely related photosynthetic taxa. In this study, we found that a cryptophyte, Cryptomonas borealis, is in a stage just prior to the loss of photosynthesis. Cryptomonas borealis was mixotrophic, possessed photosynthetic activity, and grew independent of light. The plastid genome of C. borealis had distinct features, including increases of group II introns with mobility, frequent genome rearrangements, incomplete loss of inverted repeats, and abundant small/medium/large-sized structural variants. These features provide insight into the evolutionary process leading to the loss of photosynthesis.     

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.     

Structural and drug-binding properties of alpha(1)-acid glycoprotein in reverse micelles

Alpha(1)-acid glycoprotein (AGP) is a glycoprotein that consists of 183 amino acid residues and five carbohydrate chains and binds to neutral and basic drugs. We examined the structural properties and ligand-binding capacity of AGP in interactions with reverse micelles. Also, detailed information was obtained by comparing several different states of AGP. Interaction with reverse micelles induced a unique conformational transition (beta-sheet to alpha-helices) in AGP and decreased the binding capacity for the basic drug, chlorpromazine and the steroid hormone, progesterone to AGP. These structural conformations are very similar to those observed under conditions of acidity and high ionic strength (pH 2.0, 1.5 M NaCl). This structure seems to be an intermediate between the native state and the denatured state, possibly a molten globule. The present results suggest that when AGP interacts with the biomembrane, it undergoes a structural transition to a unique structure that differs from the native and denatured states and has a reduced ligand-binding capacity.     

Probenecid-induced changes in the clearance of pranoprofen enantiomers

Probenecid is known to inhibit the elimination of several acidic drugs. Its influence on the pharmacokinetics of pranoprofen was investigated in rabbit after a single intravenous injection of racemic mixture (5 mg/kg). Levels of (-)-(R)- and (+)-(S)-pranoprofen and their glucuronide (after hydrolysis with sodium hydroxide) were determined in plasma, urine, and several tissues. The plasma concentration of the (+)-(S)-isomer was higher than that of the (-)-(R)-form. Oral coadministered probenecid (100 mg/kg) resulted in an increased plasma concentration of both enantiomers. Probenecid reduced the apparent total clearance and excretion of pranoprofen enantiomers in urine. It had a slight effect on the tissue distribution of pranoprofen at the dose used, but significantly reduced the formation of glucuronide for both enantiomers to the same extent in kidney microsomes. The differences caused by probenecid were significant with respect to its ability to inhibit glucuronidation in the kidney and subsequent excretion into urine, but enantioselective effects were negligible.     

Lipids in the heart: a source of fuel and a source of toxins

PURPOSE OF REVIEW: How do lipids arrive in the heart and other tissues? This review focuses on new information on pathways of lipid uptake into the heart. RECENT FINDINGS: Fatty acids, the major cardiac fuel, are obtained from either lipoproteins or free fatty acids associated with albumin. The heart is the tissue with the most robust expression of lipoprotein lipase, and recent data attest to the importance of this enzyme in supplying optimal amounts of fatty acids for the heart. Genetic deletion of CD36 also shows that this transporter is important for cardiac uptake of lipids. Retinoid acquisition by the heart involves pathways parallel to those used for fatty acid uptake: a pathway for acquisition of core lipoprotein retinyl ester and another for nonlipoprotein retinol. Dilated lipotoxic cardiomyopathy is the consequence of excess lipid uptake. SUMMARY: Genetic modifications that affect lipid uptake, oxidation, and storage are being exploited to elucidate the pathophysiology of cardiomyopathies and to discover how lipids relate to heart failure in humans with obesity and diabetes mellitus. This information is likely to lead to new diagnostic categories of cardiomyopathy and more pathophysiologically appropriate treatments.     

Role of pro-oxidants and antioxidants in the anti-inflammatory and apoptotic effects of curcumin (diferuloylmethane)

Extensive research within the past half-century has indicated that curcumin (diferuloylmethane), a yellow pigment in curry powder, exhibits antioxidant, anti-inflammatory, and proapoptotic activities. We investigated whether the anti-inflammatory and proapoptotic activities assigned to curcumin are mediated through its prooxidant/antioxidant mechanism. We found that TNF-mediated NF-kappaB activation was inhibited by curcumin; and glutathione reversed the inhibition. Similarly, suppression of TNF-induced AKT activation by curcumin was also abrogated by glutathione. The reducing agent also counteracted the inhibitory effects of curcumin on TNF-induced NF-kappaB-regulated antiapoptotic (Bcl-2, Bcl-xL, IAP1), proliferative (cyclin D1), and proinflammatory (COX-2, iNOS, and MMP-9) gene products. The suppression of TNF-induced AP-1 activation by curcumin was also reversed by glutathione. Also, the direct proapoptotic effects of curcumin were inhibited by glutathione and potentiated by depletion of intracellular glutathione by buthionine sulfoximine. Moreover, curcumin induced the production of reactive oxygen species and modulated intracellular GSH levels. Quenchers of hydroxyl radicals, however, were ineffective in inhibiting curcumin-mediated NF-kappaB suppression. Further, N-acetylcysteine partially reversed the effect of curcumin. Based on these results we conclude that curcumin mediates its apoptotic and anti-inflammatory activities through modulation of the redox status of the cell.     

Effect of genetic variation on the thermal stability of human serum albumin

Reversible thermal denaturation of 33 genetic variants of human serum albumin (HSA) appeared to be a two-state process when studied by circular dichroism (CD). Fourteen single-residue variants have Tm values (midpoint of denaturation) higher than, and nine have Tm values lower than, their endogenous, wild-type counterpart. Nine single-residue variants have DeltaHv values (van't Hoff enthalpy) higher than, and 14 have DeltaHv values lower than, normal albumin. All types of combinations of positive and negative DeltaTm values and Delta(DeltaHv) values were found. Good linear correlations between mutation-induced changes of alpha-helical content and Delta(DeltaHv) values, but not DeltaTm values, were found especially for the variants mutated in domains I and III. The effect of altered chain length and glycosylation on Tm and DeltaHv was also studied. For all variants, no clear relationship was found between the changes in the thermodynamic parameters and the type of substitution, changes in protein charge or hydrophobicity. However, the protein changes taking place in domain I have a rather uniform effect (almost all of the nine variants have positive DeltaTm values and negative Delta(DeltaHv) values, i.e., they denature more easily than normal albumin but they do so at a higher temperature). The present results can be of both protein chemical relevance and of clinical interest, because they could be useful when designing stable, recombinant HSAs for clinical applications.     

RNA cleavage linked with ribosomal action

Ribonuclease LS in Escherichia coli is a potential antagonist of bacteriophage T4. When T4 dmd is mutated, this RNase efficiently cleaves T4 mRNAs and leads to the silencing of late genes, thus blocking T4 growth. We previously found that, when two consecutive ochre codons were placed in the open reading frame of T4 soc, RNase LS cleaved soc mRNA at a specific site downstream of the ochre codons. Here, we demonstrate that RNase LS cleaves soc RNA at the same site even when only a single ochre codon is present or is replaced with either an amber or an opal codon. On the other hand, disruption of the Shine-Dalgarno sequence, a ribosome-binding site required for the initiation of translation, eliminates the cleavage. These results strongly suggest that RNase LS cleaves in a manner dependent on translation termination. Consistent with this suggestion, the cleavage dependency on an amber codon was considerably reduced in the presence of amber-codon-suppressing tRNA. Instead, two other cleavages that depend on translation of the region containing the target sites occurred farther downstream. Additional analysis suggests that an interaction of the ribosome with a stop codon might affect the site of cleavage by RNase LS in an mRNA molecule. This effect of the ribosome could reflect remodeling of the high-order structure of the mRNA molecule.