Structural basis for ion selectivity in potassium-selective channelrhodopsins.

KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.

Pump-like channelrhodopsins: Not just bridging the gap between ion pumps and ion channels.

Channelrhodopsins are microbial rhodopsins that work as light-gated ion channels. Their importance has become increasingly recognized due to their ability to control the membrane potential of specific cells in a light-dependent manner. This technology, termed optogenetics, has revolutionized neuroscience, and numerous channelrhodopsin variants have been isolated or engineered to expand the utility of optogenetics. Pump-like channelrhodopsins (PLCRs), one of the recently discovered channelrhodopsin subfamilies, have attracted broad attention due to their high sequence similarity to ion-pumping rhodopsins and their distinct properties, such as high light sensitivity and ion selectivity. In this review, we summarize the current understanding of the structure-function relationships of PLCRs and discuss the challenges and opportunities of channelrhodopsin research.

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine.

ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.

Differences in vulnerability to traumatic stress among patients with psychiatric disorders: One-year follow-up study after the Great East Japan Earthquake.

AIMS: The aim of this study was to evaluate differences in vulnerability to traumatic stress and the 1-year course of post-traumatic stress symptoms among patients with pre-existing psychiatric disorders after the Great East Japan Earthquake. METHODS: The Impact of Event Scale-Revised (IES-R) was used to assess post-traumatic stress symptoms in 612 patients with schizophrenic (ICD-10 F2; n = 163), mood (F3; n = 299), or neurotic disorders (F4; n = 150) at 1-4 months and again at 13-16 months after the disaster (retention rate: 68%). RESULTS: The mean IES-R total score for all diagnostic groups was 18.6 at index and 13.4 at follow up. The mean IES-R total score for patients with neurotic disorders (22.5) was significantly higher than that of patients with mood disorders (18.1) and schizophrenic disorders (15.9). At follow up, these scores decreased for all groups and inter-group differences were not observed. CONCLUSIONS: Vulnerability to traumatic stress after a disaster was most severe in patients with neurotic disorders, followed by mood disorders, and, lastly, schizophrenic disorders. This difference among the three diagnostic groups was not found 1 year after the disaster.

Clinical features of late-onset schizophrenia in Japan: comparison with early-onset cases.

BACKGROUND: Although the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders abandoned the use of the specifier 'late-onset', a considerable number of studies have reported clinical characteristics of late-onset schizophrenia. Still, only limited research has been conducted on late-onset schizophrenia, especially in Asian countries. In this epidemiological study, the clinical characteristics of late-onset schizophrenia were examined in comparison with early-onset schizophrenia. METHODS: All patients with schizophrenia admitted to the psychiatric ward of Jichi Medical University Hospital between 1 April 1993 and 31 March 2006 were divided into two groups according to age at first onset: ≥40 years (late-onset group) and <40 years (early-onset group). The sex ratio, presence or absence of depression, schizophrenia subtype, premorbid character, marital history, and employment history at first onset were compared between the two groups. RESULTS: Of the 316 patients with schizophrenia identified, 38 patients were assigned to the late-onset group and 278 patients to the early-onset group. Mean age at onset was 23.9 ± 8.2 years for all men and 28.0 ± 13.5 years for all women. The late-onset group was characterized by more women, more paranoid type, more depressive symptoms, less introverted premorbid character, better premorbid adaptation and less neuroleptics. CONCLUSION: The characteristics of late-onset schizophrenia in Japan are in line those reported previously.

[Effects of psychological distress due to the Great East Japan Earthquake, tsunami, Fukushima Nuclear Power Plant disasters on psychiatric symptoms in patients with mental disorders: observational studies in Tochigi].

BACKGROUND: The Great East Japan Earthquake and subsequent tsunami of March 11, 2011 severely damaged a widespread region of northeastern Japan. Consequently, the Fukushima Nuclear Power Plant experienced a level seven 3 reactors melted down, which released a large amount of radioactive materials into the air. Due to the structural damage and radiation leaks, the victims are facing prolonged psychological distress. METHODS: Eighty-two subjects with mental disorders who made their initial visit during the first 4 months after the earthquake and one hundred and ninety-four subjects with mental disorders who had been admitted during the first one year after the earthquake to the Jichi Medical University Hospital, which is located at the edge of the disaster-stricken region, were recruited for this study. Enrolled participants were assessed according to ICD-10. A questionnaire survey was employed to evaluate the severity of psychological distress and total amount of damage. RESULTS: The conditions of 22% of the outpatients had been worsened by the psychological distress related to the earthquake. Seven percent of the patients who had been hospitalized showed marked exacerbations due to the psychological distress associated with the disaster. COMMENTS: It is of note that the exacerbation of psychiatric symptoms due to the disaster was evident among patients with mental disorders who lived even at the edge of the disaster area (i. e., subject to an earthquake intensity of 5 upper and 150 km from the Fukushima Nuclear Power Plant). The results suggest that the close follow-up of disaster victims with mental disorders is of critical importance.

Hypoxia induces upregulation of the deoxyribonuclease I gene in the human pancreatic cancer cell line QGP-1.

We have previously demonstrated that ischemia caused by acute myocardial infarction induces an abrupt increase of serum deoxyribonuclease I (DNase I) activity. In this study, we examined whether hypoxia can affect the levels of DNase I activity and/or its transcripts in vitro. We first exposed the human pancreatic cancer cell line QGP-1, which is the first documented DNase-I-producing cell line, to hypoxia (2% O2), and found that this induced a significant increase in both the activity and transcripts of DNase I. This response was mediated by increased transcription only from exon 1a of the two alternative transcription-initiating exons utilized simultaneously in the human DNase I gene (DNASE1); exposure of QGP-1 cells to hypoxia for 24 h resulted in a 15-fold increase of DNASE1 transcripts starting from exon 1a compared with the expression level under normoxic conditions. Promoter, electrophoretic mobility shift, and chromatin immunoprecipitation assays with QGP-1 cells exposed to hypoxia or normoxia showed that the region just upstream from exon 1a was involved in this response in a hypoxia-induced factor-1-independent, but at least in a Sp1 transcription factor-dependent manner possibly through enhanced binding of Sp1 protein to the promoter. These results indicate that DNASE1 expression is upregulated by hypoxia in the cells.

Identification of novel fibronectin fragments detected specifically in juvenile urine.

Ugl-Y (young age-related urinary glycoprotein) is an age-specific protein that we have previously identified in urine from healthy subjects under 18 years of age. Isoelectric focusing analysis of Ugl-Y gives a set of three bands, Y1, Y2 and Y3, in the pH region around 3. To determine the complete structure of Ugl-Y, purified Y1 and Y2 from pediatric urine were enzymatically cleaved, and the resulting peptides were analyzed by protein sequencing and/or MALDI-TOF MS. As a result, it was demonstrated that Y1 consists of 189 amino acid residues, and is identical to the region from A723 to R911 of fibronectin, whereas Y2 consists of 181 amino acid residues, and is identical to the region from A723 to R903. Electrophoretic analysis of the lysate prepared from COS-7 cells transfected with Y1- or Y2-expressing vectors gave specific bands corresponding to Y1 or Y2, respectively, showing the validity of the sequences determined. Partial purification of pediatric serum followed by western blotting revealed that Ugl-Y is derived from plasma. Furthermore, Ugl-Y was generated by in vitro digestion of fibronectin by acid protease in extracts of osteoclast cells. These findings suggest that Ugl-Y is probably produced by degradation of fibronectin comprising bone matrix during the process of vigorous bone resorption in children and adolescents. This is the first report on the identification and characterization of juvenile-specific fibronectin fragments excreted into urine.

Susceptibility of mammalian deoxyribonucleases I (DNases I) to proteolysis by proteases and its relationships to tissue distribution: biochemical and molecular analysis of equine DNase I.

Equine (Equus caballus) deoxyribonuclease I (DNase I) was purified from the parotid gland, and its 1295-bp cDNA was cloned. The mature equine DNase I protein consisted of 260 amino acid residues. The enzymatic properties and structural aspects of the equine enzyme were closely similar to those of other mammalian DNases I. Mammalian DNases I are classified into three types--pancreatic, parotid and pancreatic-parotid-based on their tissue distribution; as equine DNase I showed the highest activity in the parotid gland, it was confirmed to be of the parotid-type. Comparison of the susceptibility of mammalian DNases I to proteolysis by proteases demonstrated a marked correlation between tissue distribution and sensitivity/resistance to proteolysis; pancreatic-type DNase I shared properties of resistance to proteolysis by trypsin and chymotrypsin, whereas parotid-type DNase I did not. In contrast, pancreatic-parotid-type DNase I exhibited resistance to proteolysis by pepsin, whereas the other enzyme types did not. However, site-directed mutagenesis analysis revealed that only a single amino acid substitution could not account for acquisition of proteolysis resistance in the mammalian DNase I family during the course of molecular evolution. These properties are compatible with adaptation of mammalian DNases I for maintaining their activity in vivo.

Association of Gln222Arg polymorphism in the deoxyribonuclease I (DNase I) gene with myocardial infarction in Japanese patients.

AIMS: We have recently reported that serum deoxyribonuclease I (DNase I) activity, which may be involved in apoptosis, increases abruptly in the early phase of acute myocardial infarction (MI) [Kawai Y, Yoshida M, Arakawa K, Kumamoto T, Morikawa N, Masamura K, Tada H, Ito S, Hoshizaki H, Oshima S, Taniguchi K, Terasawa H, Miyamori I, Kishi K, Yasuda T. Diagnostic use of serum deoxyribonuclease I activity as a novel early-phase marker in acute myocardial infarction. Circulation 2004;109:2398-2400]. Death of vascular smooth muscle cells, in part because of apoptosis, is postulated to heighten susceptibility to disruption of vulnerable plaque, resulting in onset of MI. The present study evaluated the possibility that Gln222Arg polymorphism of the DNase I gene may be one of the factors involved in predisposition to MI. METHODS AND RESULTS: We assessed 611 Japanese patients: 311 with MI and 300 with stable angina pectoris (AP). Three common phenotypes determined by two common codominant alleles, DNASE1*1 and *2, whose corresponding gene products exhibit different properties, were found in these patient groups. The prevalence of DNASE1*2 was significantly higher in patients with MI than in those with AP (0.543 vs. 0.428, P < 0.001), being confirmed by phenotyping of the second study population. Multiple logistic regression analysis showed that the odds ratio of DNASE1*2 was 1.51 [95% confidence interval (CI) 1.04-2.18]. The association of the DNASE1*2 allele with MI was statistically significant, being independent of other conventional risk factors. CONCLUSION: Our data demonstrate that Gln222Arg polymorphism in the DNase I gene is associated with MI in the Japanese patients.

Characterization of human deoxyribonuclease I gene (DNASE1) promoters reveals the utilization of two transcription-starting exons and the involvement of Sp1 in its transcriptional regulation.

Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown to be altered by physiological and/or pathological processes. However, no information is available on the regulation of DNase I gene (DNASE1) expression in vivo or in vitro. We first mapped the transcription start sites of DNASE1 in human pancreas and in the DNase I-producing human pancreatic cancer cell line QGP-1, and revealed a novel site approximately 12 kb upstream of exon 1, which was previously believed to be the single transcription-starting exon. This initiation site marks an alternative starting exon, designated 1a. Exons 1 and 1a were used simultaneously as transcription-starting exons in pancreas and QGP-1 cells. Promoter assay, EMSA and chromatin immunoprecipitation analysis with QGP-1 cells showed the promoter region of exon 1a in which the Sp1 transcription factor is specifically involved in promoter activity. This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes. Furthermore, RT-PCR analysis indicated alternative splicing of human DNASE1 pre-mRNA in pancreas and QGP-1 cells. Only two transcripts among eight alternative splicing products identified can be translated to produce intact DNase I protein. These results suggest that human DNASE1 expression is regulated through the use of alternative promoter and alternative splicing.

Human Mpv17-like protein is localized in peroxisomes and regulates expression of antioxidant enzymes.

M-LP (Mpv17-like protein) is a protein that was initially identified in mouse tissues and shows high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early-onset glomerulosclerosis [R. Iida, T. Yasuda, E. Tsubota, H. Takatsuka, M. Masuyama, T. Matsuki, K. Kishi, M-LP, Mpv17-like protein, has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene, J. Biol. Chem. 278 (2003) 6301-6306]. Here we report the identification and characterization of a human homolog of the M-LP (M-LPH) gene. The M-LPH gene is composed of four exons, extends over 14kb on chromosome 16p13.1, and is expressed as two alternatively spliced variants comprising four and three exons, respectively, which include open-reading frames encoding two distinct isoforms composed of 196 (M-LPH1) and 147 (M-LPH2) amino acids, respectively. These two variants were expressed ubiquitously in human tissues, however only M-LPH1 was detected at the protein level. Dual-color confocal analysis of COS-7 cells transfected with a green fluorescent protein-tagged M-LPH1 demonstrated that M-LPH1 is localized in peroxisomes. In order to elucidate the function of M-LPH1, we examined the mRNA levels of several enzymes involved in the metabolism of reactive oxygen species in COS-7 cells and found that transfection with M-LPH1 down-regulates expression of the plasma glutathione peroxidase and catalase genes. These results show the existence of the human homolog of M-LP and its participation in reactive oxygen species metabolism.

Chronological trend of calorie intake and the incidence of epithelial neoplasms of the large intestine during the past 30 years in Japan.

OBJECTIVES: The authors tried to elucidate the effects of food intake on the incidences of colorectal cancer and adenoma. MATERIALS AND METHODS: Large intestines obtained from a series of consecutive autopsies performed at the Tokyo Metropolitan Geriatric Hospital were scrutinized to detect colorectal neoplasms. Chronological trends and age-dependent incidences of adenoma and overt lethal cancer were checked, while cancer in adenoma were included in the category of adenoma. Data on food consumption were obtained from the 'The National Nutrition Survey in Japan' (Ministry of Health, Labor and Welfare of Japan). RESULTS: There were 405 cancers and 12,883 adenomas in the 7,091 large intestines examined. The incidences of cancer and adenoma increased with age; overt lethal cancer was found in 5.79% and adenoma in 56.79% of the general population > or =60 years in Tokyo. Calorie intake steeply increased since 1964, the year of the Olympic Games in Tokyo, until the early 1970s. Subsequently, it has continuously declined down to the level of the late 1940s, although the ingredients have changed tremendously. Chronological trends of the incidences of colorectal cancer and adenoma showed similar patterns as calorie intake, but, the influence of calorie intake on the incidences of cancer or adenoma was manifested 18 and 24 years later, respectively. CONCLUSIONS: (1) During the last 30 years, the incidences of colorectal cancer and adenoma were 5.79 and 56.79%, respectively, in individuals aged > or =60 years in Japan. (2) The chronological trends of both lesions showed a pattern similar to that of calorie intake, but, as the trend of cancer incidence preceded the course of adenoma by 6 years, adenoma is not the sole precursor lesion of overt lethal cancer.

Clinical applications of DNase I, a genetic marker already used for forensic identification.

This review primarily summarizes the clinical applications of deoxyribonuclease I (DNase I). Human DNase I exhibits polymorphism at both the protein and DNA level, and thus is potentially one of the best biochemical markers for forensic practice. Clinically, DNase I activity in serum can be used as a novel diagnostic marker for the early detection of acute myocardial infarction and transient myocardial ischemia. Furthermore, the DNase I gene is considered to be one of the susceptibility genes for gastric and colorectal carcinoma, and myocardial infarction. Over the last decade since the discovery of the utility of its genetic polymorphism for forensic purposes, research on DNase I has expanded into clinical applications.

Identification, characterization and forensic application of novel Y-STRs.

Y-chromosomal polymorphic STRs are a powerful tool for forensic and evolutionary studies. Within the last decade, a series of Y-STR systems have been developed and demonstrated to be suitable for a variety of forensic applications including sexual assault cases and paternity testing. This review describes our recent studies on novel male-specific Y-STRs, involving identification, development of a multiplex-PCR system, population study and forensic application.

Serum deoxyribonuclease I activity can be used as a sensitive marker for detection of transient myocardial ischaemia induced by percutaneous coronary intervention.

AIMS: Cardiac markers such as troponin T (c-TnT) have proven unsuitable for the detection of early and transient myocardial ischaemia. We recently reported that abrupt elevation of serum deoxyribonuclease I (DNase I) activity in the early stage of acute myocardial infarction could be used as a diagnostic marker. To evaluate whether serum DNase I could be used as a marker of early myocardial ischaemia, we investigated alterations in its levels after transient ischaemia induced during percutaneous coronary intervention (PCI). METHODS AND RESULTS: In 24 consecutive patients with stable angina undergoing elective PCI and 12 patients undergoing coronary angiography (CAG), serum samples were tested for DNase I, creatine kinase isoenzyme MB (CK-MB), and c-TnT before, soon after, and 3 and 12-24 h after completion of the procedures. Serum DNase I activity had risen significantly from baseline by 3 h after PCI in 21 of the 24 PCI patients. The mean per cent difference from baseline in serum DNase I activity 3 h after PCI was 35.9+/-37.5%. Even among the 16 PCI patients whose levels of CK-MB and c-TnT were within the normal range, 13 showed elevation of serum DNase I activity from baseline after PCI. In the CAG patient group, DNase I activity levels remained unchanged at all times after CAG. CONCLUSION: Elevation of serum DNase I activity can be used as a sensitive marker for detection of transient myocardial ischaemia.

Improvement of a multiplex PCR system for DYS441, DYS442, DYS443, DYS444 and DYS445, and a population study in 340 Japanese males.

A multiplex PCR system for five Y-STRs (DYS441, DYS442, DYS443, DYS444 and DYS445) has been improved to increase the probability of obtaining a DNA typing result from aged samples. Newly designed PCR primers for amplification of the DYS441 and DYS442 loci and optimization of PCR conditions enabled successful typing from blood and semen stains that had been stored for more than seven years at room temperature. Analysis of 340 Japanese males revealed 7, 5, 6, 5 and 4 alleles at the DYS441, DYS442, DYS443, DYS444 and DYS445 loci, respectively, yielding 122 haplotypes with a cumulative haplotype diversity of 0.97.

A novel alternative spliced Mpv17-like protein isoform localizes in cytosol and is expressed in a kidney- and adult-specific manner.

Mpv17-like protein (M-LP) has been identified as a new protein that shows high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early onset glomerulosclerosis. We previously showed that the originally identified M-LP isoform, designated M-LPL, is, like Mpv17, localized in peroxisomes, and that transfection with M-LPL up-regulates expression of the manganese superoxide dismutase (SOD2) gene [R. Iida, T. Yasuda, E. Tsubota, H. Takatsuka, M. Masuyama, T. Matsuki, K. Kishi, M-LP, Mpv17-like protein, has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene. J. Biol. Chem. 278 (2003) 6301-6306.]. We report here the identification of a novel alternative splicing product of the M-LP gene, designated M-LPS. A comparison of the genomic sequence with the cDNA sequences and an analysis of 5'-flanking regions revealed that the two isoforms are generated by alternative usage of two promoters. M-LPS consists of the C-terminal half of M-LPL (90 amino acids) and therefore lacks the peroxisome targeting signal of membrane protein that exists near the N-terminus of M-LPL. Expression of green fluorescent protein-tagged M-LPS in COS-7 cells demonstrated that M-LPS localizes in the cytosol. In mice, M-LPS is expressed exclusively in kidneys after the age of 6 weeks. Moreover, quantitative real-time PCR analysis revealed that transfection with M-LPS up-regulates expression of the SOD2 gene and down-regulates expression of the cellular glutathione peroxidase (Gpx1) and plasma glutathione peroxidase (Gpx3) genes. Taken together, these results suggest different functional attributes of the two M-LP isoforms during aging and development.

Transcriptional regulation of the human ABO histo-blood group genes is dependent on the N box upstream of the proximal promoter.

BACKGROUND: Our previous studies of the transcriptional regulation of the human ABO gene indicated that negative regulatory elements are present in the sequence just upstream from the proximal promoter. The role of the -275 to -118 region in regulation of ABO gene transcription is further characterized. STUDY DESIGN AND METHODS: Transient transfection experiments into various cells were performed with luciferase reporter plasmids carrying ABO upstream sequences, and electrophoretic mobility shift assay was carried out with a nuclear extract prepared from the human gastric cancer KATOIII cells. RESULTS: It is shown that the -202 to -118 region is involved in the negative regulation of ABO gene transcription in all cell lines examined. Transient transfection experiments in KATOIII cells with a reporter plasmid carrying mutated N box at -196 to -191 demonstrate that the N box is a negative regulatory element in the -202 to -118 sequence. Electrophoretic mobility shift assays indicate that the N box binds with a nuclear factor from KATOIII cells. CONCLUSION: These results indicate that repression of transcription from the ABO proximal promoter is partially dependent upon the N box. Therefore, reduced binding of the protein with the N box might play a direct role in ABO gene expression.

Molecular evolution of shark and other vertebrate DNases I.

We purified pancreatic deoxyribonuclease I (DNase I) from the shark Heterodontus japonicus using three-step column chromatography. Although its enzymatic properties resembled those of other vertebrate DNases I, shark DNase I was unique in being a basic protein. Full-length cDNAs encoding the DNases I of two shark species, H. japonicus and Triakis scyllia, were constructed from their total pancreatic RNAs using RACE. Nucleotide sequence analyses revealed two structural alterations unique to shark enzymes: substitution of two Cys residues at positions 101 and 104 (which are well conserved in all other vertebrate DNases I) and insertion of an additional Thr or Asn residue into an essential Ca(2+)-binding site. Site-directed mutagenesis of shark DNase I indicated that both of these alterations reduced the stability of the enzyme. When the signal sequence region of human DNase I (which has a high alpha-helical structure content) was replaced with its amphibian, fish and shark counterparts (which have low alpha-helical structure contents), the activity expressed by the chimeric mutant constructs in transfected mammalian cells was approximately half that of the wild-type enzyme. In contrast, substitution of the human signal sequence region into the amphibian, fish and shark enzymes produced higher activity compared with the wild-types. The vertebrate DNase I family may have acquired high stability and effective expression of the enzyme protein through structural alterations in both the mature protein and its signal sequence regions during molecular evolution.