39 research outputs found
Early Proton Transfer Reaction in a Primate Blue-Sensitive Visual Pigment
The proton transfer reaction belongs to one of the key
triggers
for the functional expression of membrane proteins. Rod and cone opsins
are light-sensitive G-protein-coupled receptors (GPCRs) that undergo
the cis–trans isomerization
of the retinal chromophore in response to light. The isomerization
event initiates a conformational change in the opsin protein moiety,
which propagates the downstream effector signaling. The final step
of receptor activation is the deprotonation of the retinal Schiff
base, a proton transfer reaction which has been believed to be identical
among the cone opsins. Here, we report an unexpected proton transfer
reaction occurring in the early photoreaction process of primate blue-sensitive
pigment (MB). By using low-temperature UV–visible spectroscopy,
we found that the Lumi intermediate of MB formed in transition from
the BL intermediate shows an absorption maximum in the UV region,
indicating the deprotonation of the retinal Schiff base. Comparison
of the light-induced difference FTIR spectra of Batho, BL, and Lumi
showed significant α-helical backbone C=O stretching and protonated
carboxylate C=O stretching vibrations only in the Lumi intermediate.
The transition from BL to Lumi thus involves dramatic changes in protein
environment with a proton transfer reaction between the Schiff base
and the counterion resulting in an absorption maximum in the UV region
Table_1_A short decision time for transcatheter embolization can better associate mortality in patients with pelvic fracture: a retrospective study.docx
BackgroundEarly use of hemostasis strategies, transcatheter arterial embolization (TAE) is critical in cases of pelvic injury because of the risk of hemorrhagic shock and other fatal injuries. We investigated the influence of delays in TAE administration on mortality.MethodsPatients admitted to the Advanced Critical Care Center at Gifu University with pelvic injury between January 2008 and December 2019, and who underwent acute TAE, were retrospectively enrolled. The time from when the doctor decided to administer TAE to the start of TAE (needling time) was defined as “decision-TAE time.”ResultsWe included 158 patients, of whom 23 patients died. The median decision-TAE time was 59.5 min. Kaplan–Meier curves for overall survival were compared between patients with decision-TAE time above and below the median cutoff value; survival was significantly better for patients with values below the median cutoff value (p = 0.020). Multivariable Cox proportional hazards regression analysis revealed that the longer the decision-TAE time, the higher the risk of mortality (p = 0.031). TAE duration modified the association between decision-TAE time and overall survival (p = 0.109), as shorter TAE duration (procedure time) was associated with the best survival rate (p for interaction = 0.109).ConclusionDecision-TAE time may play a key role in establishing resuscitation procedures in patients with pelvic fracture, and efforts to shorten this time should be pursued.</p
Tysnd1 Deficiency in Mice Interferes with the Peroxisomal Localization of PTS2 Enzymes, Causing Lipid Metabolic Abnormalities and Male Infertility
<div><p>Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids β-oxidation pathway. To clarify the <em>in vivo</em> functions and physiological role of Tysnd1, we analyzed the phenotype of <em>Tysnd1<sup>−/−</sup></em> mice. Male <em>Tysnd1<sup>−/−</sup></em> mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. <em>Tysnd1<sup>−/−</sup></em> mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of <em>Tysnd1</em> interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.</p> </div
Male <i>Tysnd1</i><sup>−/−</sup> mice are infertile.
<p>A. Semi-thin (8–10 micron) testes sections of 20 weeks old <i>Tysnd1</i><sup>+/+</sup> and <i>Tysnd1<sup>−/−</sup></i> mice were stained with hematoxylin-eosin. Abnormal, round sperm heads are visible in the seminiferous tubules of <i>Tysnd1</i><sup>−/−</sup> mice. B. Abnormal morphology of <i>Tysnd1</i><sup>−/−</sup> sperms. Epididymal sperms of 15 weeks-old <i>Tysnd1</i><sup>+/+</sup> and <i>Tysnd1</i><sup>−/−</sup> mice were stained with mitochondrial stains (MitoFluor Red) and nuclear staining (DAPI). Scale bar = 20 µm. C. Percentage of sperms showing normal morphology in <i>Tysnd1</i><sup>+/+</sup> (n = 4), <i>Tysnd1</i><sup>+/−</sup> (n = 4) and <i>Tysnd1</i><sup>−/−</sup> (n = 5) mice. Each error bar represents the mean ± SE. D. Anti-MN9 antibody immunostaining and Hoechst nuclear staining of epididymal sperms isolated from a 20 weeks old <i>Tysnd1</i><sup>−/−</sup> mouse (red: acrosome and blue: nucleus). Arrow heads and arrows indicate abnormal round-headed sperms and normal sperms, respectively. Scale bar = 5 µm. E. Acrosomes of a semi-thin testis section from a 10 weeks old <i>Tysnd1</i><sup>−/−</sup> and a heterozygous control mouse were stained with PNA-FITC (green) and Hoechst nuclear stain (blue). F. EM image of a <i>Tysnd1</i><sup>−/−</sup> caudal epididymal sperm. The round-headed sperm lacks the acrosome and shows an abnormal mitochondrial sheath (M) around the nucleus (N). Scale bar = 1 µm. G. EM image showing normal spermatogenesis in <i>Tysnd1</i><sup>+/−</sup> male mice. S7: step 7 round spermatid; S16: step 16 elongated spermatid. Acrosomes (A) are normally formed. Scale bar = 2 µm. H. EM image of <i>Tysnd1</i><sup>−/−</sup> elongated spermatid. S16: step 16 spermatid. In some spermatids the acrosome (*) is detached from the nucleus (N). Scale bar = 2 µm.</p
Generation of <i>Tysnd1</i><sup>−/−</sup> mice.
<p>A. Map of <i>Tysnd1</i><sup>−/−</sup> targeting constructs. P1, P2 and P3 indicate primers used for genotyping by PCR. B. Identification of genotyping by PCR. Wild type and <i>Tysnd1</i><sup>−/−</sup> genotype were identified by 237 bp and 339 bp PCR products, respectively. C. Relative expression level of <i>Tysnd1</i> mRNA measured by quantitative real-time PCR in liver. D. Tysnd1 protein was absent in the liver of <i>Tysnd1</i><sup>−/−</sup> mice as shown by Western blotting using anti-Tysnd1 antibody. E. The expression of known Tysnd1 substrates in the liver homogenate was detected by Western blotting using anti-Acox1, -ScpX/Scp2, -Hsd17b4 and -Acaa1 antibodies. Processed forms of Tysnd1 substrates were not detected in <i>Tysnd1</i><sup>−/−</sup> mice. Arrows indicate the processed form of each enzyme. F. Peroxisomal β-oxidation activity was measured by [1-C<sup>14</sup>]lignoceric acids in 15 weeks old control diet-fed (CE2, Clea Japan) male mice liver homogenate. ***<i>p</i><0.001. Each error bar represents the mean ± SE in <i>n</i> = 3.</p
Tysnd1 processes Agps and Phyh <i>in vitro</i>.
<p>A. COS7 cells were transiently co-transfected with Agps-V5 and Tysnd1 expression plasmids. With increasing amounts of Tysnd1 unprocessed Agps-V5 decreased (arrow). Processed Agps-V5 is indicated by an arrowhead B. Agps processing by Tysnd1 is specific and was affected by MG132 proteasome inhibitor. C. Western blot of testes extract using anti-Agps antibody shows unprocessed (arrow) and processed (arrowhead) forms of Agps. D. COS7 cells were transiently co-transfected with Phyh-V5 and Tysnd1 expression plasmids. With increasing amounts of Tysnd1, unprocessed Phyh-V5 decreased (arrow) and processed Phyh-V5 increased (arrowhead). E. Western blot of liver extract using anti-Phyh antibody shows unprocessed (arrow) and processed (arrowhead) forms of Phyh.</p
Ten-week-old male <i>Tysnd1</i><sup>−/−</sup> mice accumulate phytanic acid.
<p>A. Livers after 13 days of phytol feeding. Left: enlarged, beige-coloured liver of phytol-fed <i>Tysnd1</i><sup>−/−</sup> mouse. Right: liver of phytol-fed <i>Tysnd1</i><sup>+/+</sup> mouse. B. Hematoxylin-eosin stained semi-thin liver sections. Lipid droplets are indicated by yellow arrow heads. Green arrow heads indicate giant cells. C. Blood serum-derived phytanic acid measurement by GC/MS normalized for C16∶0 (hexadecanoic acid) content. Each error bar represents the mean ± SE in n = 4–9. ***<i>p</i><0.001. D. Measurement of blood serum C24∶0 (tetracosanoic acid), C25∶0 (pentacosanoic acid) and C26∶0 (hexacosanoic acid) VLCFAs by GC/MS normalized by C22∶0 (docosanoic acid) content. Each error bars: mean ± SE in n = 4–9. ***<i>p</i><0.001. E. Measurement of liver peroxisomal β-oxidation activity with or without orally administered 0.5% phytol in carboxyl methyl cellulose (CMC). Each error bars: mean ± SE in <i>n</i> = 3–6. NS: not significant. ***<i>p</i><0.001. F. EM image of liver sections with or without orally administered phytol in CMC. The blue and purple arrow heads indicate peroxisomes and autophagosomes, respectively.Bar: 2 µm. G. Numbers of peroxisomes and autophagosomes counted within same field areas of EM images. Error bars: mean ± SE in n = 5–8. ***<i>p</i><0.001.</p
Proposed model of PTS2-protein import into peroxisomes in presence or absence of Tysnd1.
<p>A. Unprocessed PTS2 protein is imported from the cytoplasm into the peroxisome by Pex7 in association with the long isoform of Pex5 (Pex5pL). Tysnd1 processes the imported protein after the PTS2 signal and Pex7 and Pex5pL are recycled to the cytoplasm. The PTS2-containing fragment is exported from the peroxisome to the cytoplasm. Subsequent Pex5 and Pex7 docking components Pex13 and Pex14 are displayed as gray circles, but not individually labelled for reasons of simplicity. B. Tysnd1-deficient mouse peroxisome. Unprocessed PTS2-containing proteins remain bound as Pex7-Pex5pL cargo complex. Saturation of Pex7 and Pex5pL transport with relative shortage of free Pex5pL and Pex7 causes partial peroxisomal location appearance and accumulation of peroxisomal proteins outside the peroxisome.</p
A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies
<div><p>Mitochondrial disorders have the highest incidence among congenital metabolic disorders characterized by biochemical respiratory chain complex deficiencies. It occurs at a rate of 1 in 5,000 births, and has phenotypic and genetic heterogeneity. Mutations in about 1,500 nuclear encoded mitochondrial proteins may cause mitochondrial dysfunction of energy production and mitochondrial disorders. More than 250 genes that cause mitochondrial disorders have been reported to date. However exact genetic diagnosis for patients still remained largely unknown. To reveal this heterogeneity, we performed comprehensive genomic analyses for 142 patients with childhood-onset mitochondrial respiratory chain complex deficiencies. The approach includes whole mtDNA and exome analyses using high-throughput sequencing, and chromosomal aberration analyses using high-density oligonucleotide arrays. We identified 37 novel mutations in known mitochondrial disease genes and 3 mitochondria-related genes (<i>MRPS23</i>, <i>QRSL1</i>, and <i>PNPLA4</i>) as novel causative genes. We also identified 2 genes known to cause monogenic diseases (<i>MECP2</i> and <i>TNNI3</i>) and 3 chromosomal aberrations (6q24.3-q25.1, 17p12, and 22q11.21) as causes in this cohort. Our approaches enhance the ability to identify pathogenic gene mutations in patients with biochemically defined mitochondrial respiratory chain complex deficiencies in clinical settings. They also underscore clinical and genetic heterogeneity and will improve patient care of this complex disorder.</p></div
Breakdown of 142 patients categorized by the type of mutations/variants in comprehensive genomic analysis.
<p>Numerals in each colored box indicate the number of patients. Patients harboring multiple variants were assigned to each box on the basis of the highest priority variant in all cases.</p