Identification of a region of the tobacco mosaic virus 126- and 183-kilodalton replication proteins which binds specifically to the viral 3'-terminal tRNA-like structure

UV irradiation of a mixture of an isolated tobacco mosaic virus (TMV; tomato strain L [TMV-L]) RNA-dependent RNA polymerase complex and the TMV-L RNA 3'-terminal region (3'-TR) resulted in cross-linking of the TMV-L 126-kDa replication protein to the TMV-L 3'-TR. Using both Escherichia coli-expressed proteins corresponding to parts of the 126-kDa protein and mutants of the 3'-TR, the interacting sites were located to a 110-amino-acid region just downstream of the core methyltransferase domain in the protein and a region comprising the central core C and domain D2 in the 3'-TR. Mutation to alanine of a tyrosine residue at position 409 or a tyrosine residue at position 416 in the protein binding region abolished cross-linking to the 3'-TR, and corresponding mutations introduced into TMV-L RNA abolished its ability to replicate in tomato protoplasts, with no detectable production of either plus- or minus-strand RNA. The results are compatible with a model for initiation of TMV-L minus-strand RNA synthesis in which an internal region of the TMV-L 126-kDa protein first binds to the central core C and domain D2 region of the TMV-L 3'-TR and is then followed by binding of the 183-kDa protein to this complex and positioning of the catalytically active site of the polymerase domain close to the 3'-terminal CCCA initiation site.     

Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein

ATPase activity associated with P-glycoprotein (Pgp) is characterized by three drug-dependent phases: basal (no drug), drug-activated, and drug-inhibited. To understand the communication between drug-binding sites and ATP hydrolytic sites, we performed steady-state thermodynamic analyses of ATP hydrolysis in the presence and absence of transport substrates. We used purified human Pgp (ABCB1, MDR1) expressed in Saccharomyces cerevisiae (Figler, R. A., Omote, H., Nakamoto, R. K., and Al-Shawi, M. K. (2000) Arch. Biochem. Biophys. 376, 34-46) as well as Chinese hamster Pgp (PGP1). Between 23 and 35 degrees C, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis of saturating MgATP in the presence of saturating drug concentrations (kcat), from which we calculated the intrinsic enthalpic, entropic, and free energy terms for the rate-limiting transition states. Linearity of the Arrhenius plots indicated that the same rate-limiting step was being measured over the temperature range employed. Using linear free energy analysis, two distinct transition states were found: one associated with uncoupled basal activity and the other with coupled drug transport activity. We concluded that basal ATPase activity associated with Pgp is not a consequence of transport of an endogenous lipid or other endogenous substrates. Rather, it is an intrinsic mechanistic property of the enzyme. We also found that rapidly transported substrates bound tighter to the transition state and required fewer conformational alterations by the enzyme to achieve the coupling transition state. The overall rate-limiting step of Pgp during transport is a carrier reorientation step. Furthermore, Pgp is optimized to transport drugs out of cells at high rates at the expense of coupling efficiency. The drug inhibition phase was associated with low affinity drug-binding sites. These results are consistent with an expanded version of the alternating catalytic site drug transport model (Senior, A. E., Al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289). A new kinetic model of drug transport is presented.     

Melatonin and N-acetylcysteine have beneficial effects during hepatic ischemia and reperfusion

This study was designed to study the effects of Melatonin (Mel) and N-Acetylcystein (NAC) on hepatic ischemia/reperfusion (I/R) injury in rats. For this purpose Wistar albino rats were subjected to 45 minutes of hepatic ischemia followed by 60 minutes of reperfusion period. Melatonin (10 mg/kg) or NAC (150 mg/kg) were administered alone or in combination, intraperitoneally, 15 minutes prior to ischemia and just before reperfusion. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined to assess liver functions. Liver tissues were taken for determination of malondialdehyde (MDA) levels, an end product of lipid peroxidation; glutathione (GSH) levels, a key antioxidant; protein carbonyl concentration (protein oxidation) (PO), a specific marker of oxidative damage of proteins; and myeloperoxidase (MPO) activity, as an indirect index of neutrophil infiltration. Plasma ALT and AST activities were higher in ischemia/reperfusion group than in control. They were decreased in the groups given Mel, NAC or the combination. Hepatic GSH levels, significantly depressed by I/R, were elevated to control levels in the combination group, whereas treatment with Mel or NAC alone provided only a limited protection. Hepatic MDA and PO levels, and MPO activity were significantly increased by I/R. The increase in these parameters were partially decreased by Mel or NAC alone, whereas treatment with the combination reduced these values back to control levels. In conclusion, considering the dosages used, Mel appeared to be significantly more potent than NAC in reversing the oxidative damage induced by I/R. Our findings show that Mel and NAC have beneficial effects against the I/R injury and due to their synergistic effects, when administered in combination, may have a more pronounced protective effects on the liver.     

Melatonin ameliorates ionizing radiation-induced oxidative organ damage in rats

This study was designed to study the effects of the potential radioprotective properties of pharmacological doses of melatonin against organ damage induced by whole-body irradiation (IR) in rats. A total of 32 male Sprague-Dawley rats were exposed to irradiation performed with a LINAC producing 6 MV photons at a focus 100 cm distant from the skin. Under ketamine anaesthesia, each rat received a single whole-body dose of 800 cGy. Immediately before and after IR, rats were treated with either saline or melatonin (20 mg/kg and 10 mg/kg, i.p.) and decapitated at 12-h after exposure to irradiation. Another group of rats was followed for 72-h after IR, where melatonin (10 mg/kg, i.p.) injections were repeated once daily. Tissue levels of malondialdehyde (MDA)--an index of lipid peroxidation--, glutathione (GSH)--a key to antioxidant--and myeloperoxidase (MPO) activity--an index of neutrophil infiltration--were estimated in liver, lung, colon and intestinal tissues. The results demonstrate that both 12-h and 72-h following IR, tissue levels of MDA were elevated (p<0.05-0.001), while GSH levels were reduced (p<0.05-0.001) in all organs. On the other hand, melatonin, reduced the levels of MDA and increased the GSH levels significantly, (p<0.05-0.001). MPO activity was increased significantly in the colonic tissue at the both 12-h and 72-h, and in the hepatic tissue at the 72-h following IR, which were reduced by melatonin (p<0.01-0.001). In the lung tissue enzyme activity was decreased at 72nd h of post-irradiation. In conclusion, the increase in MDA levels and MPO activity and the concomitant decrease in GSH levels demonstrate the role of oxidative mechanisms in irradiation-induced tissue damage, and melatonin, by its free radical scavenging and antioxidant properties, ameliorates irradiation-induced organ injury. Thus, supplementing cancer patients with adjuvant therapy of melatonin may have some benefit for successful radiotherapy.     

Characterization of two antagonistic strains of<Emphasis Type="Italic">Rahnella aquatilis</Emphasis> isolated from soil in egypt

In an attempt to obtain biological control agents for controlling bacterial spot of cucumber, over 250 bacterial strains were isolated from agricultural soil samples, collected from different localities in Giza Governorate (Egypt) and screened for in vitro antibiosis towards Xanthomonas campestris. Only 2 strains showed antagonistic activity. They and their culture filtrates restricted the growth of a number of G- and G(+)-indicator bacteria. On Chrome Azurol S agar, both strains exhibited a marked siderophore production. Biolog plates identified these strains as Rahnella aquatilis. Their characteristics were studied and compared with literature data on R. aquatilis. This antagonistic bacterium has not been previously isolated in Egypt.     

Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4

The blockage of replication forks can result in the disassembly of the replicative apparatus and reversal of the fork to form a DNA junction that must be processed in order for replication to restart and sister chromatids to segregate at mitosis. Fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4 are endonucleases that have been implicated in the processing of aberrant DNA junctions formed at stalled replication forks. Here we have investigated the activity of purified Mus81-Eme1 and Mus81-Mms4 on substrates that resemble DNA junctions that are expected to form when a replication fork reverses. Both enzymes cleave Holliday junctions and substrates that resemble normal replication forks poorly or not at all. However, forks where the equivalents of either both the leading and lagging strands or just the lagging strand are juxtaposed at the junction point, or where either the leading or lagging strand has been unwound to produce a fork with a single-stranded tail, are cleaved well. Cleavage sites map predominantly between 3 and 6 bp 5' of the junction point. For most substrates the leading strand template is cleaved. The sole exception is a fork with a 5' single-stranded tail, which is cleaved in the lagging strand template.     

Reduction of alpha-Gal expression by relocalizing alpha-galactosidase to the trans-Golgi network and cell surface

Historically, the most effective means of modifying cell surface carbohydrates has required the intracellular overexpression of glycosyltransferases or glycosidases and is dependent on the enzymes occupying a cellular localization close to the carbohydrate structures they modify. We report on relocalizing the lysosomal resident glycosidase human alpha-galactosidase to other regions of the cell, Golgi and cell surface, where it is in closer proximity for cleaving the carbohydrate structure Galalpha(1,3)Gal. Relocalization of alpha-galactosidase was achieved by using the transmembrane and cytoplasmic domains from the human protein furin, which is known to localize in the trans-Golgi network (TGN) and cell surface. Two chimeric forms of alpha-galactosidase were generated, one directing it to the TGN of the cell and the other to the cell surface, as shown by confocal microscopy. The relocalized enzymes have the ability to cleave terminal alpha-galactose as detected by expression on the cell surface. Furthermore, when expressed as a transgene in mice, the TGN form of alpha-galactosidase was more effective at decreasing cell surface terminal alpha-galactose than was the native lysosomal form. When expressed in conjunction with the alpha1,2fucosyltransferase that also decreases Galalpha(1,3)Gal, the reduction was additive. The ability to relocalize enzymes that modify cell surface carbohydrate structures has far-reaching implications in biology and may be useful in such fields as xenotransplantation and treatment of glycosidase disorders.     

Oligosaccharide and sucrose complexes of amylosucrase. Structural implications for the polymerase activity

The glucosyltransferase amylosucrase is structurally quite similar to the hydrolase alpha-amylase. How this switch in functionality is achieved is an important and fundamental question. The inactive E328Q amylosucrase variant has been co-crystallized with maltoheptaose, and the structure was determined by x-ray crystallography to 2.2 A resolution, revealing a maltoheptaose binding site in the B'-domain somewhat distant from the active site. Additional soaking of these crystals with maltoheptaose resulted in replacement of Tris in the active site with maltoheptaose, allowing the mapping of the -1 to +5 binding subsites. Crystals of amylosucrase were soaked with sucrose at different concentrations. The structures at approximately 2.1 A resolution revealed three new binding sites of different affinity. The highest affinity binding site is close to the active site but is not in the previously identified substrate access channel. Allosteric regulation seems necessary to facilitate access from this binding site. The structures show the pivotal role of the B'-domain in the transferase reaction. Based on these observations, an extension of the hydrolase reaction mechanism valid for this enzyme can be proposed. In this mechanism, the glycogen-like polymer is bound in the widest access channel to the active site. The polymer binding introduces structural changes that allow sucrose to migrate from its binding site into the active site and displace the polymer.