Purification and characterization of an (ADP-ribose)n glycohydrolase from human erythrocytes

An (ADP-ribose)n glycohydrolase from human erythrocytes was purified approximately 13,000-fold and characterized. On sodium dodecyl sulfate/polyacrylamide gel the purified enzyme appeared homogeneous and had an estimated relative molecular mass (Mr) of 59,000. Amino acid analysis showed that the enzyme had a relatively high content of acidic amino acid residues and low content of basic amino acid residues. Isoelectrofocusing showed that the enzyme was an acidic protein with pI value of 5.9. The mode of hydrolysis of (ADP-ribose)n by this enzyme was exoglycosidic, yielding ADP-ribose as the final product. The Km value for (ADP-ribose)n (average chain length, n = 15) was 5.8 microM and the maximal velocity of its hydrolysis was 21 mumol.min-1.mg protein-1. The optimum pH for enzyme activity was 7.4 KCl was more inhibitory than NaCl. The enzyme activity was inhibited by ADP-ribose and cAMP but not the dibutyryl-derivative (Bt2-cAMP), cGMP or AMP. These physical and catalytic properties are similar to those of cytosolic (ADP-ribose)n glycohydrolase II, but not to those of nuclear (ADP-ribose)n glycohydrolase I purified from guinea pig liver [Tanuma, S., Kawashima, K. & Endo, H. (1986) J. Biol. Chem. 261, 965-969]. Thus, human erythrocytes contain (ADP-ribose)n glycohydrolase II. The kinetics of degradation of poly(ADP-ribose) bound to histone H1 by purified erythrocyte (ADP-ribose)n glycohydrolase was essentially the same as that of the corresponding free poly(ADP-ribose). In contrast, the glycohydrolase showed appreciable activity of free oligo(ADP-ribose), much less activity on the corresponding oligo(ADP-ribose) bound to histone H1. The enzyme had more activity on oligo(ADP-ribose) bound to mitochondrial and cytosolic free mRNA ribonucleoprotein particle (mRNP) proteins than on oligo(ADP-ribose) bound to histone H1. It did not degrade mono(ADP-ribosyl)-stimulatory guanine-nucleotide-binding protein (Gs) and -inhibitory guanine-nucleotide-binding protein (Gi) prepared with cholera and pertussis toxins, respectively. These results suggest that cytosolic (ADP-ribose)n glycohydrolase II may be involved in extranuclear de(ADP-ribosyl)n-ation, but not in membrane de-mono(ADP-ribosyl)ation.     

Identification and characterization of canine growth differentiation factor-9 and its splicing variant

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor-β (TGF-β) superfamily, is expressed exclusively in the oocyte within the ovary and plays essential roles in the ovarian function in mammals. However, a possible involvement of GDF-9 in canine ovarian physiology that has a unique ovulation process among mammals has not been studied. Interestingly, we have isolated two types of cDNA clones generated by an alternative splicing from a canine ovarian total RNA. The predominant long form cDNA shares a common precursor structure with GDF-9s in other species whereas the minor short form cDNA has a 172 amino acid truncation in the proregion. Using a transient expression system, we found that the long form cDNA has a defect in mature protein production whereas the short form cDNA readily produces mature protein. However, mutations at one or two N-glycosylation sites in the mature domain of the short form GDF-9 caused a loss in mature protein production. These results suggest that the prodomain and N-linked glycosylation of the mature domain regulate proper processing and secretion of canine GDF-9. Based on the biological functions of GDF-9, these characteristics of canine GDF-9 could be causatively linked to the unique ovulation process in the Canidae.     

Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.     

High prevalence of sulfadoxine/pyrimethamine resistance alleles in Plasmodium falciparum parasites from Bangladesh

In Bangladesh, despite the official introduction of artemisinin combination therapy in 2004, chloroquine+sulfadoxine/pyrimethamine has been used for the treatment of uncomplicated malaria. To assess the distribution of pfcrt, pfmdr1, dhfr, and dhps genotypes in Plasmodium falciparum, we conducted hospital- and community-based surveys in Bandarban, Bangladesh (near the border with Myanmar) in 2007 and 2008. Using nested PCR followed by digestion, 139 P. falciparum isolates were genotyped. We found fixation of a mutation at position 76 in pfcrt and low prevalence of a mutation at position 86 in pfmdr1. In dhfr, the highest pyrimethamine resistant genotype quadruple mutant was found in 19% of isolates, which is significantly higher prevalence than reported in a previous study in Khagrachari (1%) in 2002. Microsatellite haplotypes flanking dhfr of the quadruple mutants in Bangladesh were identical or very similar to those found in Thailand and Cambodia, indicating a common origin for the mutant in these countries. These observations suggest that the higher prevalence of the dhfr quadruple mutant in Bandarban is because of parasite migration from Myanmar. However, continuous use of sulfadoxine/pyrimethamine would have also played a role through selection for the dhfr quadruple mutant. These results indicate an urgent need to collect molecular epidemiological information regarding dhfr and dhps genes, and a review of current sulfadoxine/pyrimethamine usage with the aim of avoiding the widespread distribution of high levels of resistant parasites in Bangladesh.     

The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins

Ricin is a potent cytotoxic protein derived from the higher plant Ricinus communis that inactivates eukaryotic ribosomes. In this paper we have studied the mechanism of action of ricin A-chain on rat liver ribosomes in vitro. Our findings indicate that the toxin inactivates the ribosomes by modifying both or either of two nucleoside residues, G4323 and A4324, in 28 S rRNA. These nucleotides are located close to the alpha-sarcin cleavage site and become resistant to all ribonucleases tested. The examination of the lability of phosphodiester bonds of these nucleotides to both mild alkaline digestion and aniline treatment at acidic pH suggests that the base of A4324 is removed by the toxin. This unique activity of ricin A-chain was also observed when naked 28 S rRNA is used as a substrate, indicating that the toxin directly acts on the RNA. Similar activity on 28 S rRNA is also exhibited by abrin and modeccin, ricin-related toxins, suggesting a general mechanistic pathway for ribosome inactivation by lectin toxins.     

The mechanism of action of barley toxin: a type 1 ribosome-inactivating protein with RNA N-glycosidase activity

In a previous report (Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130) it was shown that the RNA N-glycosidase activity of ricin A-chain was responsible for the ability of this protein to inactivate eukaryotic ribosomes. The objective of the present study was to determine whether a similar mechanism was used by a ribosome-inactivating protein from pearled barley (barley toxin). Rat liver ribosomes were incubated either with ricin A-chain or barley toxin, and the rRNA was extracted and treated with acidic aniline to hydrolyze phosphodiester bonds rendered susceptible by removal of a purine or pyrimidine base. Evaluation of the rRNA by polyacrylamide/agarose electrophoresis disclosed two 28 S rRNA-derived fragments which differed in size from those generated by untreated (control) ribosomes. Sequencing of the smaller of these fragments confirmed that - as is the case for ricin A-chain - the aniline-sensitive site in barley toxin-treated ribosomes was between A and G in 28 S rRNA. We conclude that barley toxin inactivates ribosomes via a mechanism identical to that of ricin A-chain: enzymatic hydrolysis of the N-glycosidic bond at A of 28 S rRNA.     

Effects of angiotensin I-converting enzyme inhibitor, SQ 14225, in nomal men.

Effects of an orally active angiotensin I-converting enzyme inhibitor, SQ 14225, on the actions of angiotensin I (AI) infused intravenously for 120 to 390 min were studied in 5 normal men. When 20 ng/kg/min of AI infusion was started immediately after a single oral administration of 100 mg of SQ 14225, a significant rise in blood pressure (BP) was observed for the first 15 min, but BP began to fall from 17 min and returned to the pretreatment level at 45 min. This BP level continued at least to 120 min and in one subject to 180 min. In this subject BP began to rise again from 185 min and reached the level of 15 min at 390 min. Plasma AI level increased gradually from 45 min. At 15 min plasma renin activity (PRA) decreased and plasma aldosterone (PA) increased, but then PRA began to increase and PA began to decrease. At 120 min the values of PRA and PA were similar to the pretreatment values. In one subject plasma AI and PRA began to decrease and PA began to increase after 120 or 180 min. On the other hand, in the 5 men sole AI infusion caused a continued BP rise, PRA decrease and PA increase, and sole SQ 14225 administration caused increases in plasma AI and PRA and a decrease in PA but no BP change. From these results it was concluded that complete blockade and partial inhibition of AI conversion by 100 mg of oral SQ 14225 lasted for about 2.5 and 6.5 hr, respectively and that BP rise, PRA suppression and aldosterone stimulation after AI infusion were entirely due to the actions of angiotensin II converted from AI.     

A Highlights from MBoC Selection: Dual Functions of Yeast tRNA Ligase in the Unfolded Protein Response: Unconventional Cytoplasmic Splicing of HAC1 Pre-mRNA Is Not Sufficient to Release Translational Attenuation

The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1^u mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1ⁱ mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1ⁱ mRNA. Furthermore, the HAC1 5′ UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1^u mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.     

Human-Induced Trophic Cascades along the Fecal Detritus Pathway

Human presence and activity in tropical forest is thought to exert top-down regulation over the various ‘green-world’ pathways of plant-based foodwebs. However, these effects have never been explored for the ‘brown-world’ pathways of fecal-detritus webs. The strong effects of humans on tropical game mammals are likely to indirectly influence fecal detritivores (including Scarabaeine dung beetles), with subsequent indirect impacts on detrivore-mediated and plant-facilitating detrital processes. Across a 380-km gradient of human influence in the western Brazilian Amazon, we conducted the first landscape-level assessment of human-induced cascade effects on the fecal detritus pathway, by coupling data on human impact, game mammal and detritivore community structure, and rate measurements of a key detritus process (i.e. dung beetle-mediated secondary seed dispersal). We found evidence that human impact indirectly influences both the diversity and biomass of fecal detritivores, but not detritivore-mediated processes. Cascade strength varied across detritivore groups defined by species'' traits. We found smaller-bodied dung beetles were at higher risk of local decline in areas of human presence, and that body size was a better predictor of cascade structure than fecal resource manipulation strategy. Cascade strength was also stronger in upland, unflooded forests, than in seasonally flooded forests. Our results suggest that the impact of human activity in tropical forest on fecal-detritus food web structure is mediated by both species'' traits and habitat type. Further research will be required to determine the conditions under which these cascade effects influence fecal-detritus web function.     

Two genes controlling acute phase responses by the antitumor polysaccharide,lentinan

Lentinan, a -1,6;1,3-glucan, is tumor-specific for transplantable mouse solid-type tumors and it also stimulates the production of acute phase proteins (APPs). The APP response to lentinan is of the delayed type (DT-APR) and differs from that to lipopolysaccharide, which is acute. We found that the responses were genetically controlled in mice and that low responsiveness is dominant (Maeda et al. 1991). Using 123 segregants of crosses between SWR/J (a high responder) andMus spretus (a low responder), we analyzed the linkage between DT-APR responsiveness and the DNA polymerase chain reaction-simple sequence lenght polymorphism (PCR-SSLP) phenotype using 80 chromosome-specific microsatellite markers. We identified two loci (ltn1.1 andltn1.2) responsible for DT-APR.ltn1.1 is closely linked toD3Mit11 on chromosome 3 andltn1.2 toD11Nds9 on chromosome 11 (P<0.001). The linkage analysis also suggested thatltn1.2 is the major determinant for DT-APR. Correlation between lentinan-specific IL-6 mRNA expression (the late expression) controlled recessively and DT-APR induction suggests that theltn1 loci control some process(es) of IL-6 expression in the regulation step before NF-IL6.