Mutant alpha-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin

Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.     

Msn2p/Msn4p-activation is essential for the recovery from freezing stress in yeast

Recent data suggest that the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subtype plays a pivotal role in the pathogenesis of effective disorders and in the action of antidepressant drugs. After chronic treatment with the antidepressants desipramine or paroxetine, we measured by immunoprecipitation and Western blotting, the changes in the interaction of AMPA receptor subunits with proteins involved in trafficking and/or stabilization of the subunits into synaptic membranes of the hippocampus. Both antidepressants increased the interaction of GluR1 subunit with stargazin and of GluR2/3 with NSF. Paroxetine increased the interaction of GluR1 with Rab4A, and desipramine markedly increased the interaction of GluR1 with SAP97. Paroxetine, but not desipramine, also increased membrane levels of CaMKII, autophosphorylated CaMKII and GluR1 phosphorylated at the CaMKII site. Interactions of GluR1 and GluR2/3 with proteins implicated in AMPA receptor trafficking and with scaffolding proteins appear to account for the enhanced membrane expression of AMPA receptors in the hippocampus after antidepressant treatment.     

Concerning the dynamic instability of actin homolog ParM

Apoptosis is a highly conserved procedure of cell death and occurs under various stimuli, including oxidative stress. A small heat shock protein, alphaB-crystallin, is found to process resistance to apoptosis in some cells and tissues. But the mechanisms under this protective role are not fully understood. In the present study, we reported the early protective role of alphaB-crystallin against hydrogen peroxide-induced apoptosis in mice myogenic C(2)C(12) cells. alphaB-Crystallin interacted with p53, a proapoptotic protein, during cell apoptosis and such protein interaction mainly occurred in the cytoplasm of the cells, suggesting that the interaction of alphaB-crystallin with p53 might prevent the translocation of p53 from cytoplasm to mitochondria. Hence, this study provides a hint that alphaB-crystallin affects on p53 mitochondrial translocation during oxidative stress-induced apoptosis.     

IRS-1 transgenic mice show increased epididymal fat mass and insulin resistance

Insulin receptor substrate-1 (IRS-1) is the major substrate of both the insulin receptor and the IGF-1 receptor. In this study, we created IRS-1 transgenic (IRS-1-Tg) mice which express human IRS-1 cDNA under control of the mouse IRS-1 gene promoter. In the IRS-1-Tg mice, IRS-1 mRNA expression was significantly increased in almost all tissues, but its protein expression was increased in very limited tissues (epididymal fat and skeletal muscle). IRS-1-Tg mice showed glucose intolerance and significantly enlarged epididymal fat mass, as well as elevated serum TNF-α concentrations. Importantly insulin signaling was significantly attenuated in the liver of IRS-1-Tg mice, which may contribute to the glucose intolerance. Our results suggest that excess IRS-1 expression may not provide a beneficial impact on glucose homeostasis in vivo.     

Enhanced activity of ventricular Na+-HCO3- cotransport in pressure overload hypertrophy

The Na(+)-HCO(3)(-) cotransporter (NBC) plays a key role in intracellular pH (pH(i)) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pH(i) was measured in single cells using the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)5-(6)carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC (J(NBC)) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na(+)/H(+) exchange was also increased in hypertrophied myocytes, the relative enhancement of J(NBC) was larger, 6) membrane depolarization markedly increased J(NBC) in hypertrophied myocytes, and 7) losartan, an ANG II AT(1) receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na(+) overload, which may render the ventricles more vulnerable to Ca(2+) overload during ischemia-reperfusion.     

Molecular and morphological characterization of Leveillula (Ascomycota: Erysiphales) on monocotyledonous plants

Leveillula on monocotyledonous plants have been recorded as L. taurica by several authors, whereas the fungus on Allium has been described as an independent species, namely L. allii, by some authors. We sequenced ca 600 bp of the rDNA ITS region for two Leveillula specimens from Allium and Polianthes (both from monocotyledons) and compared them with several already published sequences from Leveillula isolates from dicotyledons. Pair-wise percentages of sequence divergences were calculated for all Leveillula isolates. The ITS sequence of the Polianthes isolate was identical to L. taurica on Helianthus and Vicia. The sequence of the Allium isolate was 99.5 % identical to L. taurica on Euphorbia, Haplophylum, Peganum, etc. These results suggest close relationships between monocot and dicot pathogenic Leveillula species. The identity between two monocot isolates was 98.4 %. Phylogenetic analysis revealed that the two monocot isolates do not group into a clade together. This result suggests that Leveillula acquired parasitism to monocots at least twice independently.     

The intramembrane active site of GlpG, an E. coli rhomboid protease, is accessible to water and hydrolyses an extramembrane peptide bond of substrates

Escherichia coli GlpG is an orthologue of the rhomboid proteases that catalyse intramembrane proteolysis of specific membrane proteins. We previously showed that it can cleave a type I model membrane protein, Bla-LY2-MBP, having the second transmembrane region of lactose permease (LY2) in vivo and in vitro at the predicted periplasm-membrane boundary region of LY2. Here we investigated the environment of the active site regions of GlpG in the membrane-integrated state by examining the modifiability of Cys residues introduced into the regions around the catalytic residues with membrane-permeable and -impermeable alkylating reagents. The results indicate that the enzyme active site is fully open to the external aqueous phase. GlpG also cleaved a similar fusion protein, Bla-GknTM-MBP, having the transmembrane region of Gurken (GknTM), a physiological substrate of Drosophila rhomboids. Engineered Cys residues in the cleavage site regions of the LY2 and GknTM sequences were efficiently modified with a membrane-impermeable alkylating reagent, showing that these regions are exposed to the periplasm. These results suggest that GlpG cleaves an extramembrane region of substrates, unlike the currently prevailing view that this class of membrane proteases acts against a membrane-embedded polypeptide segment after its lateral entrance into the enzyme's active site.     

Boss/Sev signaling from germline to soma restricts germline-stem-cell-niche formation in the anterior region of Drosophila male gonads

Drosophila germline stem cells are regulated by the somatic microenvironment, or "niche," which ensures that the stem cells can both self-renew and produce functional gametes throughout adult life. However, despite its prime importance, little is known about how niche formation is regulated during gonadal development. Here, we demonstrate that a receptor tyrosine kinase, Sevenless (Sev), is required to ensure that the niche develops in the anterior region of the male embryonic gonads. Sev is expressed in somatic cells within the posterior region of the gonads. Sev is activated by a ligand, Bride of sevenless (Boss), which is expressed by the germline, to prevent ectopic niche differentiation in the posterior gonadal somatic cells. Thus, we propose that signal transduction from germline to soma restricts expansion of the germline-stem-cell niche in the gonads.     

Immunogenicity and safety of an inactivated quadrivalent influenza vaccine in healthy adults: a phase II,open‐label,uncontrolled trial in Japan

Two antigenically distinct B strain lineages of influenza virus have co‐circulated since the mid‐1980s; however, inactivated trivalent influenza vaccines contain only one B lineage. The mismatch between the circulating and vaccine lineages has been a worldwide issue. In this study, an inactivated quadrivalent influenza vaccine (QIV) candidate containing two B lineages was manufactured and its immunogenicity and safety evaluated in an open‐label, uncontrolled trial. In this phase II trial, 50 subjects aged 20–64 years received two doses of QIV s.c. 1 to 4 weeks apart. Sera were collected pre‐ and post‐vaccination and safety assessed from the first vaccination to 21 ± 7 days after the second vaccination. After the first vaccination, hemagglutination inhibition titers against each strain increased markedly; the seroconversion rate, geometric mean titer ratio and seroprotection rate being 94.0%, 24.93, and 100.0%, respectively, for the A/H1N1pdm09 strain; 94.0%, 12.47, and 98.0%, respectively, for the A/H3N2 strain; 54.0%, 4.99, and 66.0%, respectively, for B/Yamagata strain, and 72.0%, 6.23 and 80.0%, respectively, for the B/Victoria strain, thus fulfilling the criteria of the European Medical Agency's Committee for Medicinal Products for Human Use. Also, the QIV induced sufficient single radial hemolysis and neutralizing antibodies against all four vaccine strains. No noteworthy adverse events were noted. The results of this trial demonstrate that QIV is well tolerated and immunogenic for each strain, suggesting that QIV potentially improves protection against influenza B by resolving the issue of B lineage mismatch.     

The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses

DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage.