[Effects of Coarse and Fine Atmospheric Particulate Matter on a Mast Cell Line].

We investigated the cytotoxicity on a mast cell line (C57 cells) of water-soluble extracts of coarse (>3 µm, PM>3) and fine (0.05-3 µm, PM0.05-3) atmospheric particulates collected from April 2016 to March 2019 in Fukuoka, Japan. We examined the direct cytotoxicity with punched-out membrane filter fragments of PM>3 and PM0.05-3 collected from April 2019 to March 2021, without extraction of the components. Also, cell proliferation and degranulation assays were conducted under conditions which caused no cytotoxicity with water-soluble extracts of PM>3 from FY2016 and PM>3 direct samples from FY2019. The findings revealed the significant direct cytotoxicity of many PM>3 and all PM0.05-3 samples, with higher cytotoxicity for PM0.05-3 (FY2019-2020). These results were different from the cytotoxicity effects of water-soluble extracts of PM>3 and PM0.05-3 samples (FY2016) in previous studies. In addition, inhibition of cell proliferation and induction of degranulation were significantly induced in a few PM>3 samples, showing a correlation with the suspended particulate matter (SPM) concentrations. This method using punched-out membrane filters is convenient and useful for assessing the direct effects of atmospheric particles on a small scale.

Cytotoxic Effects of Water-Soluble Extracts of Coarse and Fine Atmospheric Particulate Matter on Mast Cell Lines.

Fine particulate matter (PM2.5) pollution causes serious health disorders, because PM2.5 becomes deposited in the tracheobronchial and alveoli regions. In the extrathoracic region, there are more deposits of coarse particulate matter than fine particulates. As adverse health issues caused by coarse particulates have not been well investigated, this study examined the cytotoxicity of water-soluble extracts of both fine (0.05-3 µm, PM0.05-3) and coarse (> 3 µm, PM>3) particulates collected from April 2016 to March 2019 in Fukuoka, Japan. Also evaluated were concentrations of NH4+ and SO42-, multi-components of well-known secondary generation substances. The findings revealed that PM>3 showed stronger cytotoxic effects on mast cell lines than PM0.05-3. Cytotoxic effects were observed at concentrations of over 15 mM of (NH4)2SO4 and over 30 mM of NH4Cl. In contrast, Na2SO4 caused few cytotoxic effects up to a concentration of 50 mM. The causative substances for this cytotoxicity may not have been NH4+ and SO42- because their PM>3 concentrations indicating the largest cytotoxic effects were 1 and 0.4 mM, respectively. The cytotoxicities of PM>3 and PM0.05-3 were the highest in the first half of FY2016. These cytotoxicities seem to be due to cross-border pollution, although this pollution has been declining in recent years. An increasing trend of cytotoxicity was observed in the second half of FY2018. This study showed that cytotoxicity and particulate concentrations are not always correlated. Thus, we should focus not only on the quantity of atmospheric particulate matter, but also on its quality.

Slowly progressive d-bifunctional protein deficiency with survival to adulthood diagnosed by whole-exome sequencing.

d-Bifunctional protein (DBP) deficiency is an autosomal recessive disorder of peroxisomal fatty acid oxidation caused by mutations in HSD17B4. It is typically fatal by the age of two years with symptom onset during the neonatal period, and survival until late childhood is rare. We herein report the case of a patient with DBP deficiency surviving until adulthood, who showed severe sensorineural deafness, disturbances in language acquisition, slowly progressive cerebellar ataxia, and peripheral neuropathy. This patient, in whom findings of prior investigations were nondiagnostic, had been followed up as having an early-onset spinocerebellar degeneration of unknown etiology. Whole-exome sequencing analysis at the age of 36 showed two heterozygous variants in the gene HSD17B4, which encodes DBP in this patient. A panel of peroxisomal investigations showed normal levels of very long chain fatty acids (VLCFAs) in plasma and elevated serum phytanic acid levels. Recently, an increasing number of patients with DBP deficiency surviving until adolescence/adulthood have been reported, in whom abnormalities in the levels of VLCFAs and other peroxisomal metabolites are marginal or nonexistent. Genetic analysis of HSD17B4 should be considered in adult patients with cerebellar ataxia, peripheral neuropathy, and pyramidal signs in addition to sensorineural auditory disturbance since childhood.

The Biosynthesis of the Pyrimidine Moiety of Thiamin in Halobacterium salinarum.

The biosynthetic pathway of the pyrimidine moiety of thiamin was studied in the archaean Halobacterium salinarum. Thiamin is biosynthesized from 4-amino-5-hydroxymethyl-2-methylpyrimidine (pyrimidine) and 5-(2-hydroxyethyl)-4-methylthiazole (thiazole). The pyrimidine and the thiazole are biosynthesized de novo in microorganisms. The biosynthetic routes of pyrimidine in microorganisms differ between eukaryote and eubacteria. In the eukaryote Saccharomyces cerevisiae, histidine and pyridoxine are the precursors of pyrimidine, while in the eubacterium Escherichia coli, pyrimidine is biosynthesized from 5-aminoimidazole ribonucleotide (AIR), an intermediate of purine biosynthesis. Tracer investigations revealed that [(15)N]-, [1-(13)C]- and [2-(13)C] glycine, precursors of AIR, were incorporated into the pyrimidine in H. salinarum. These results suggested that the biosynthetic route of the pyrimidine in H. salinarum is similar to that of E. coli.

The Biosynthesis of the Thiazole Moiety of Thiamin in the Archaeon Halobacterium salinarum.

The biosynthetic pathways of the thiazole moiety of thiamin were studied in the archaeon Halobacterium salinarum. Thiamin is generated by the union of 4-amino-5-hydroxymethyl-2-methylpyrimidine (pyrimidine) and 5-(2-hydroxyethyl)-4-methylthiazole (thiazole). The biosynthesis of thiazole is different in facultative anaerobes, aerobes and eukaryotes. In eukaryotes, the C-4, -4', -5, -5' and -5" of the thiazole is biosynthesized from nicotinamide adenine dinucleotide (NAD), with cysteine as S donor and the C-2 and N atoms of glycine. In facultative anaerobic bacteria, such as Escherichia coli, the precursors of the thiazole are the N and C-2 atoms from tyrosine and C-4, -4', -5, -5' and -5" from 1-deoxy-D-xylurose-5-phosphate, again with cysteine as S donor. In aerobic bacteria, such as Bacillus subtilis, L-tyrosine is replaced by glycine. In Archaea, known as the third domain of life, the biosynthetic pathway of thiamin has not yet been elucidated. In the present study in the archaeon H. salinarum, it was shown that both the N and C-2 from glycine are incorporated into the thiazole, rather than the N atom coming from L-tyrosine. These results show that thiazole biosynthesis in H. salinarum more closely resembles the biosynthetic pathway found in eukaryotes.

The direct precursor of the pyrimidine moiety of thiamin is not urocanic acid but histidine in Saccharomyces cerevisiae.

The biosynthetic route of the pyrimidine moiety of thiamin is different in prokaryotes and eukaryotes. In prokaryotes, the pyrimidine moiety is synthesized from aminoimidazole ribonucleotide, an intermediate of purine biosynthesis, while in eukaryotes, we have reported that the N-1, C-2, and N-3 atoms of the imidazole ring of histidine are incorporated into N-3, C-4, and the amino group attached to the C-4 atoms of the pyrimidine moiety, respectively, as a unit; the rest of the atoms of the pyrimidine moiety originate from pyridoxine as a unit. It has been reported that urocanic acid, the deaminated compound of histidine, is the direct precursor of the pyrimidine moiety. In the present report, we have investigated whether histidine or urocanic acid is the direct precursor of the pyrimidine moiety in Saccharomyces cerevisiae, using tracer experiments with 1) (13)C-formate and urocanic acid, 2) (15)N-NH(4)Cl and urocanic acid, 3) (15)N-NH(4)Cl and histidine, and 4) (13)C-histidine and urocanic acid. The GC-MS analysis revealed that the incorporation of the (15)N atom of (15)NH(4)Cl was not affected by the presence of urocanic acid, although it was affected by histidine, and the incorporation of (13)C-histidine was not affected by the presence of urocanic acid. These results confirm that histidine is the direct precursor of the pyrimidine moiety of thiamin in S. cerevisiae.

Effect of L-serine on the biosynthesis of aromatic amino acids in Escherichia coli.

It is well known that some amino acids inhibit bacterial growth. L-Serine is known to inhibit the growth of Escherichia coli by inhibition of homoserine dehydrogenase (EC 1.1.1.3). It has been reported that this L-serine inhibition may be prevented by the addition of L-isoleucine or L-threonine to the medium. In our study, however, recovery of the growth inhibition of Escherichia coli by L-serine occurred in the presence of several amino acids, especially L-phenylalanine. In an attempt to further elucidate this inhibition mechanism, different intermediates of aromatic amino acid biosynthesis were added to the growth medium. Recovery from the inhibition did not occur in the presence of prephenate but did occur when phenylpyruvate was added to the medium. The specific activity of prephenate dehydratase decreased in cells grown in the presence of L-serine. However. L-serine did not inhibit in vitro prephenate dehydratase activity, and the expression of pheA, which encodes the prephenate dehydratase, was not depressed by L-serine. We suggest that L-serine acts via another inhibition mechanism. Although this inhibition mechanism has not been fully elucidated, our results suggest that the addition of L-serine to the growth medium inhibits prephenate dehydratase synthesis and thus affects L-phenylalanine biosynthesis.

Statin-induced apoptosis linked with membrane farnesylated Ras small G protein depletion, rather than geranylated Rho protein.

Rhabdomyolysis is a severe adverse effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins). This myopathy is strongly enhanced by the combination with statins and fibrates, another hypolipidaemic agent. We have evaluated the initial step of statin-induced apoptosis by the detection of membrane flip-flop using flow cytometric analysis. L6 rat myoblasts were treated with various statins (atorvastatin (3 microM), cerivastatin (3 microM), fluvastatin (3 microM), pravastatin (3 mM), or simvastatin (3 microM)) for 2, 4 or 6 h followed by reacting with FITC-conjugated annexin V for the detection of initial apoptosis signal (flip-flop). Various statin-treated myoblasts were significantly stained with FITC-annexin V at 6 h, whereas they were not detected at 2 h. Moreover, immunoblot analysis indicated that when the cells were treated with cerivastatin (3 microM), membrane-associated Ras protein was activated and detached until 6 h, resulting in cell death through the consequent activation of caspase-8. On the other hand, since cytosolic Ras activation did not activate, there is still an unknown mechanism in statin-related Ras depletion. In conclusion, statin-induced apoptosis in muscular tissue was directly initiated by the farnesyl-anchored Ras protein depletion from cell membrane with subsequent apoptosis.

[An estimation chart for the possibility of aspiration in patients with severe motor and intellectual disabilities: its reliability and accuracy].

Although the most common cause of death in patients with severe motor and intellectual disabilities (SMID) is aspiration pneumonia, there are no criteria to detect aspiration. We have been making an evaluation tool to detect aspiration in patients with SMID easily, in cooperation with the project of comprehensive study of disability, social health, and welfare, supported by the Ministry of Health, Labor and Welfare, Japan. Here we studied the reliability and accuracy of the 2001 edition of the "questionnaires for functions of eating and swallowing" and "estimation chart for the possibility of aspiration" in 20 patients with risk of aspiration (13 with cerebral palsy, six with acquired cerebral palsy and one with muscle disease). Family members or nursing staffs taking care of them answered the questionnaires. A pediatrician and an occupational therapist or speech therapist checked the chart. Reliability was checked by the agreement between the estimation of the two professionals, and accuracy was determined by comparing the results of the chart and videofluolography. The results showed good reliability (kappa 0.63) and accuracy (kappa 0.47). Based on these results and additional analysis of the check items, the new edition of the chart was established.

[Effect of intrapulmonary percussive ventilation in a severely disabled patient with persistent pulmonary consolidation].

An intrapulmonary percussive ventilator (IPV) improves airway clearance and lung function, and is useful for wide variety of respiratory disorders, such as cystic fibrosis, chronic obstructive pulmonary disease, aspiration pneumonia, and neuromuscular diseases. However, there are few reports on IPV use in patients with severe neurological impairment, scoliosis and thoracic deformity. They have poor mobility of the rib cage and difficulty in sputum expectoration. The use of IPV significantly improved persistent consolidation shown by chest computed tomography (CT) in one of such patients. The patient was a 33-year-old woman with severe spastic quadriplegia and tracheostomy and she was dependent on mechanical ventilation because of chronic restrictive respiratory failure. After fever and mild hypoxemia for one day, chest CT revealed consolidation of the left lower lobe. An IPV-I ventilator was used for 15 min once a week, with a stroke frequency of 250-300 cycles/min and pressure of 22 PSI. Mechanical ventilation was withheld during the IPV therapy. Chest physiotherapy was also done. According to the worsening of the consolidation on chest CT, the frequency of IPV was changed to once a day at day 23 and then to twice a day. Chest CT at day 44 showed further improvement. In patients with severe motor and intellectual disabilities, it is sometimes difficult to control progressive deterioration of pulmonary function and persistent atelectasis even with tracheostomy, mechanical ventilation, and conventional physiotherapy. Our results indicate that IPV may improve respiratory functio and the quality of life in such patients.

Evaluation of the synergistic adverse effects of concomitant therapy with statins and fibrates on rhabdomyolysis.

Rhabdomyolysis is a severe adverse effect of hypolipidaemic agents such as statins and fibrates. We evaluated this muscular cytotoxicity with an in-vitro culture system. Cellular apoptosis was determined using phase-contrast and fluorescein microscopic observation with Hoechst 33342 staining. L6 rat myoblasts were treated with various statins and bezafibrate under various conditions. With statins only, skeletal cytotoxicity was ranked as cerivastatin > fluvastatin > simvastatin > atorvastatin > pravastatin in order of decreasing potency. Combined application of fibrates enhanced atorvastatin-induced myopathy, which causes little apoptosis alone. These results suggest that statins and fibrates synergistically aggravate rhabdomyolysis.

[Adverse effects of dichloroacetate in a girl with mitochondrial disorder].

We present the effects and adverse effects of dichloroacetate (DCA) in a girl with mitochondrial disorder. Oral administration of DCA 50 mg/kg per day, reduced the elevated levels of lactate to below the normal range. Treatment with DCA ameliorated electroencephalogram abnormalities, but caused the adverse effects with hepatomegaly and decreased activity, which were improved by reduction or withdrawal of DCA. The decreased activity may be an adverse effect on the central nervous system. The dosage of DCA should be adjusted for each patient.