Generation of Rodent Malaria Parasites with a High Mutation Rate by Destructing Proofreading Activity of DNA Polymerase δ

Plasmodium falciparum malaria imposes a serious public health concern throughout the tropics. Although genetic tools are principally important to fully investigate malaria parasites, currently available forward and reverse tools are fairly limited. It is expected that parasites with a high mutation rate can readily acquire novel phenotypes/traits; however, they remain an untapped tool for malaria biology. Here, we generated a mutator malaria parasite (hereinafter called a ‘malaria mutator’), using site-directed mutagenesis and gene transfection techniques. A mutator Plasmodium berghei line with a defective proofreading 3′ → 5′ exonuclease activity in DNA polymerase δ (referred to as PbMut) and a control P. berghei line with wild-type DNA polymerase δ (referred to as PbCtl) were maintained by weekly passage in ddY mice for 122 weeks. High-throughput genome sequencing analysis revealed that two PbMut lines had 175–178 mutations and a 86- to 90-fold higher mutation rate than that of a PbCtl line. PbMut, PbCtl, and their parent strain, PbWT, showed similar course of infection. Interestingly, PbMut lost the ability to form gametocytes during serial passages. We believe that the malaria mutator system could provide a novel and useful tool to investigate malaria biology.     

Differential expression of semaphorin 3A and its receptors during mouse retinal development

Semaphorins not only function in axon guidance during development but also contribute to various other biological processes. We have now examined the expression of semaphorin 3A (Sema3A) and its receptor components neuropilin 1 (Npn1) and plexin A (PlxA) during development of the mouse retina. Immunohistofluorescence analysis revealed that the expression patterns of Sema3A and Npn1 were similar during embryonic and postnatal development. The expression pattern of PlxA was also similar to those of Sema3A and Npn1 during embryonic and early postnatal (before eye opening) developments. However, the pattern of PlxA expression changed markedly after eye opening, with the expression disappearing from the optic nerve and increasing in intensity in the retinal pigment epithelium. Immunoprecipitation analysis showed that Sema3A interacted with PlxA in the retinal pigment epithelial cell line ARPE19 but not in the retinal ganglion cell line RGC5, whereas the opposite pattern of association was apparent for Sema3A and Npn1. Given that atmospheric oxygen is thought to play a role in the differentiation and maintenance of various ocular cell types, our results suggest that Sema3A-PlxA signalling activated by an effect of ambient oxygen on PlxA expression may contribute to differentiation of the retinal pigment epithelium. Copyright ? 2012 John Wiley & Sons, Ltd.     

Filament Formation of the FtsZ/Tubulin-like Protein TubZ from the Bacillus cereus pXO1 Plasmid

Stable maintenance of low-copy-number plasmids requires partition (par) systems that consist of a nucleotide hydrolase, a DNA-binding protein, and a cis-acting DNA-binding site. The FtsZ/tubulin-like GTPase TubZ was identified as a partitioning factor of the virulence plasmids pBtoxis and pXO1 in Bacillus thuringiensis and Bacillus anthracis, respectively. TubZ exhibits high GTPase activity and assembles into polymers both in vivo and in vitro, and its “treadmilling” movement is required for plasmid stability in the cell. To investigate the molecular mechanism of pXO1 plasmid segregation by TubZ filaments, we determined the crystal structures of Bacillus cereus TubZ in apo-, GDP-, and guanosine 5′-3-O-(thio)triphosphate (GTPγS)-bound forms at resolutions of 2.1, 1.9, and 3.3 Å, respectively. Interestingly, the slowly hydrolyzable GTP analog GTPγS was hydrolyzed to GDP in the crystal. In the post-GTP hydrolysis state, GDP-bound B. cereus TubZ forms a dimer by the head-to-tail association of individual subunits in the asymmetric unit, which is similar to the protofilament formation of FtsZ and B. thuringiensis TubZ. However, the M loop interacts with the nucleotide-binding site of the adjacent subunit and stabilizes the filament structure in a different manner, which indicates that the molecular assembly of the TubZ-related par systems is not stringently conserved. Furthermore, we show that the C-terminal tail of TubZ is required for association with the DNA-binding protein TubR. Using a combination of crystallography, site-directed mutagenesis, and biochemical analysis, our results provide the structural basis of the TubZ polymer that may drive DNA segregation.     

Protease Activity of Procaspase-8 Is Essential for Cell Survival by Inhibiting Both Apoptotic and Nonapoptotic Cell Death Dependent on Receptor-interacting Protein Kinase 1 (RIP1) and RIP3

Caspase-8 has an important role as an initiator caspase during death receptor-mediated apoptosis. Moreover, it has been reported to contribute to the regulation of cell fate in various types of cells including T-cells. In this report, we show that caspase-8 has an essential role in cell survival in mouse T-lymphoma-derived L5178Y cells. The knockdown of caspase-8 expression decreased the growth rate and increased cell death, both of which were induced by the absence of protease activity of procaspase-8. The cell death was associated with reactive oxygen species (ROS) accumulation, caspase activation, and autophagosome formation. The cell death was inhibited completely by treatment with ROS scavengers, but only partly by treatment with caspase inhibitors, expression of Bcl-xL, and knockdown of caspase-3 or Atg-7 which completely inhibits apoptosis or autophagosome formation, respectively, indicating that apoptosis and autophagy-associated cell death are induced simultaneously by the knockdown of caspase-8 expression. Further analysis indicated that RIP1 and RIP3 regulate this multiple cell death, because the cell death as well as ROS production was completely inhibited by not only treatment with the RIP1 inhibitor necrostatin-1, but also by knockdown of RIP3. Thus, in the absence of protease activity of procaspase-8, RIP1 and RIP3 simultaneously induce not only nonapoptotic cell death conceivably including autophagic cell death and necroptosis but also apoptosis through ROS production in mouse T-lymphoma cells.     

Effects of spring-killed pine trees on the epidemics of pine wilt disease

Pine wilt disease (PWD) is caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, and is transmitted by cerambycid beetles. In some pine trees infected with the PWNs in Japan, foliage changes from green to brown in summer to autumn of a nematode infection year (summer- autumn-killed trees) and the others in the following spring of a nematode infection year (spring-killed trees). The vector beetles require 1 or 2?years for development in cool summer areas and 1?year in warm summer areas. To evaluate the effects of the spring-killed trees and vectors with a long developmental time on the PWD epidemics, we presented simple mechanistic mathematical models. The models showed that it was possible for spring-killed trees to cause PWD epidemics when the transmission rate was high, and the efficacy of spring-killed trees as infection source was similar to that of summer?C autumn-killed trees. Spring-killed trees and vector beetles with a developmental time of 2?years harbored in summer- autumn-killed trees delayed epidemic timing by 3?C10?years or actually suppressed epidemics.     

The triple helical structure and stability of collagen model peptide with 4(S)-hydroxyprolyl-Pro-Gly units

Extensive studies on the structure of collagen have revealed that the hydroxylation of Pro residues in a variety of model peptides with the typical (X-Y-Gly)(n) repeats (X and Y: Pro and its analogues) represents one of the major factors influencing the stability of triple helices. While(2S,4R)-hydroxyproline (Hyp) at the position Y stabilizes the triple helix, (2S,4S)-hydroxyproline (hyp) at the X-position destabilizes the helix as demonstrated that the triple helix of (hyp-Pro-Gly)(15) is less stable than that of (Pro-Pro-Gly)(15) and that a shorter peptide (hyp-Pro-Gly)(10) does not form the helix. To clarify the role of the hydroxyl group of Pro residues to play in the stabilization mechanism of the collagen triple helix, we synthesized and crystallized a model peptide (Pro-Hyp-Gly)(4) -(hyp-Pro-Gly)(2) -(Pro-Hyp-Gly)(4) and analyzed its structure by X-ray crystallography and CD spectroscopy. In the crystal, the main-chain of this peptide forms a typical collagen like triple helix. The majority of hyp residues take down pucker with exceptionally shallow angles probably to relieve steric hindrance, but the remainders protrude the hydroxyl group toward solvent with the less favorable up pucker to fit in a triple helix. There is no indication of the existence of an intra-molecular hydrogen bond between the hydroxyl moiety and the carbonyl oxygen of hyp supposed to destabilize the triple helix. We also compared the conformational energies of up and down packers of the pyrrolidine ring in Ac-hyp-NMe(2) by quantum mechanical calculations.     

A shift in sphingolipid composition from C24 to C16 increases susceptibility to apoptosis in HeLa cells

Sphingolipids, major lipid components of the eukaryotic plasma membrane, have a variety of physiological functions and have been associated with many diseases. They have also been implicated in apoptosis. Sphingolipids are heterogeneous in their acyl chain length, with long-chain (C16) and very long-chain (C24) sphingolipids being predominant in most mammalian tissues. We demonstrate that knockdown of ELOVL1 or CERS2, which catalyze synthesis of C24 acyl-CoAs and C24 ceramide, respectively, drastically reduced C24 sphingolipid levels with a complementary increase in C16 sphingolipids. Under ELOVL1 or CERS2 knockdown conditions, cisplatin-induced apoptosis significantly increased. Enhanced sensitivity to cisplatin-induced apoptosis exhibited close correlation with increases in caspase-3/7 activity. No significant alterations in sphingolipid metabolism such as ceramide generation were apparent with the cisplatin-induced apoptosis, and inhibitors of ceramide generation had no effect on the apoptosis. Apoptosis induced by UV radiation or C6 ceramides also increased in ELOVL1 or CERS2 knockdown cells. Changes in the composition of sphingolipid chain length may affect susceptibility to stimuli-induced apoptosis by affecting the properties of cell membranes, such as lipid microdomain/raft formation.     

The effect of water loading on the urinary ratio of cortisone to cortisol in healthy subjects and a new approach to the evaluation of the ratio as an index for in vivo human 11β-hydroxysteroid dehydrogenase 2 activity

Factors that give rise to a large variation in the urinary ratio of free cortisone to cortisol (UFE/UFF) were investigated to accurately estimate 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity in humans in vivo. A water loading test was first carried out in two healthy subjects to examine the effect of water intake or urine volume on the urinary ratio of free cortisone to cortisol (UFE/UFF). The ratio was found to increase by water loading. We also examined urinary concentrations and amounts of cortisol, cortisone, creatinine, Na(+), K(+), and Cl(-), and urine volume, as possible factors affecting the urinary ratio (UFE/UFF), in 60 urine samples obtained from 15 healthy volunteers. Among these factors tested, the urinary concentration of cortisol was most highly correlated with the UFE/UFF ratio (r=-0.858), indicating that the in vivo activity of 11β-HSD2 (UFE/UFF) should fluctuate with the changes of the urinary concentration of cortisol. Based on the findings, we proposed a new estimation method of in vivo activity of 11β-HSD2 in humans, using the UFE/UFF ratio correlated with the urinary concentration of cortisol (UFE/UFF-cortisol concentration). Taking into consideration the intra-individual variabilities in the urinary concentration of cortisol, there were no significant within-day variations in 11β-HSD2 activity. The findings indicate that 11β-HSD2 activities can be accurately evaluated by simply measuring free cortisol and cortisone concentrations in spot urine samples. Furthermore, administrations of glycyrrhetinic acid in three healthy volunteers were performed to confirm the usefulness of the present assessment for the activity of 11β-HSD2.     

Up-regulated L-type high voltage-gated calcium channels cause increase in diazepam binding inhibitor induced by sustained morphine exposure in mouse cerebrocortical neurons

Mechanisms of increase in diazepam binding inhibitor (DBI) mRNA expression in mouse cerebrocortical neurons after sustained morphine exposure were investigated. Increases in DBI and its mRNA expressions induced by sustained morphine (0.3 μM) exposure for 3 days were completely abolished by naloxone and nifedipine, but not by ω-agatoxin VIA and ω-conotoxin GIVA. Increase in [³H]diltiazem binding to the particulate fractions from the morphine-treated neurons was due to increased B_max value with no changes in K_d value. Western blot analysis on l-type high voltage-gated calcium channel (HVCC) subunits revealed the increased expressions of α1C, α1D, and α2/δ1 subunits and decreased of β4 subunit expression, whereas expression of N- and P/Q-type HVCC subunits was not changed. These results indicate that morphine-induced increase in DBI mRNA expression is mediated via increased Ca²⁺ entry through up-regulated l-type HVCCs.