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1.  The p38 MAPK and JNK Pathways Protect Host Cells against Clostridium perfringens Beta-Toxin 
Infection and Immunity  2013;81(10):3703-3708.
Clostridium perfringens beta-toxin is an important agent of necrotic enteritis and enterotoxemia. Beta-toxin is a pore-forming toxin (PFT) that causes cytotoxicity. Two mitogen-activated protein kinase (MAPK) pathways (p38 and c-Jun N-terminal kinase [JNK]-like) provide cellular defense against various stresses. To investigate the role of the MAPK pathways in the toxic effect of beta-toxin, we examined cytotoxicity in five cell lines. Beta-toxin induced cytotoxicity in cells in the following order: THP-1 = U937 > HL-60 > BALL-1 = MOLT-4. In THP-1 cells, beta-toxin formed oligomers on lipid rafts in membranes and induced the efflux of K+ from THP-1 cells in a dose- and time-dependent manner. The phosphorylation of p38 MAPK and JNK occurred in response to an attack by beta-toxin. p38 MAPK (SB203580) and JNK (SP600125) inhibitors enhanced toxin-induced cell death. Incubation in K+-free medium intensified p38 MAPK activation and cell death induced by the toxin, while incubation in K+-high medium prevented those effects. While streptolysin O (SLO) reportedly activates p38 MAPK via reactive oxygen species (ROS), we showed that this pathway did not play a major role in p38 phosphorylation in beta-toxin-treated cells. Therefore, we propose that beta-toxin induces activation of the MAPK pathway to promote host cell survival.
PMCID: PMC3811753  PMID: 23876806
2.  SLC25A13 Gene Analysis in Citrin Deficiency: Sixteen Novel Mutations in East Asian Patients, and the Mutation Distribution in a Large Pediatric Cohort in China 
PLoS ONE  2013;8(9):e74544.
The human SLC25A13 gene encodes citrin, the liver-type mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), and SLC25A13 mutations cause citrin deficiency (CD), a disease entity that encompasses different age-dependant clinical phenotypes such as Adult-onset Citrullinemia Type II (CTLN2) and Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD). The analyses of SLC25A13 gene and its protein/mRNA products remain reliable tools for the definitive diagnoses of CD patients, and so far, the SLC25A13 mutation spectrum in Chinese CD patients has not been well-characterized yet.
Methods and Results
By means of direct DNA sequencing, cDNA cloning and SNP analyses, 16 novel pathogenic mutations, including 9 missense, 4 nonsense, 1 splice-site, 1 deletion and 1 large transposal insertion IVS4ins6kb (GenBank accession number KF425758), were identified in CTLN2 or NICCD patients from China, Japan and Malaysia, respectively, making the SLC25A13 variations worldwide reach the total number of 81. A large NICCD cohort of 116 Chinese cases was also established, and the 4 high-frequency mutations contributed a much larger proportion of the mutated alleles in the patients from south China than in those from the north (χ2 = 14.93, P<0.01), with the latitude of 30°N as the geographic dividing line in mainland China.
This paper further enriched the SLC25A13 variation spectrum worldwide, and formed a substantial contribution to the in-depth understanding of the genotypic feature of Chinese CD patients.
PMCID: PMC3777997  PMID: 24069319
3.  Intracellular Trafficking of Clostridium perfringens Iota-Toxin b 
Infection and Immunity  2012;80(10):3410-3416.
Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca2+ concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes.
PMCID: PMC3457585  PMID: 22825447
4.  Elucidation of the RNA Recognition Code for Pentatricopeptide Repeat Proteins Involved in Organelle RNA Editing in Plants 
PLoS ONE  2013;8(3):e57286.
Pentatricopeptide repeat (PPR) proteins are eukaryotic RNA-binding proteins that are commonly found in plants. Organelle transcript processing and stability are mediated by PPR proteins in a gene-specific manner through recognition by tandem arrays of degenerate 35-amino-acid repeating units, the PPR motifs. However, the sequence-specific RNA recognition mechanism of the PPR protein remains largely unknown. Here, we show the principle underlying RNA recognition for PPR proteins involved in RNA editing. The distance between the PPR-RNA alignment and the editable C was shown to be conserved. Amino acid variation at 3 particular positions within the motif determined recognition of a specific RNA in a programmable manner, with a 1-motif to 1-nucleotide correspondence, with no gap sequence. Data from the decoded nucleotide frequencies for these 3 amino acids were used to assign accurate interacting sites to several PPR proteins for RNA editing and to predict the target site for an uncharacterized PPR protein.
PMCID: PMC3589468  PMID: 23472078
5.  Biochemical characteristics of neonatal cholestasis induced by citrin deficiency 
AIM: To explore differences in biochemical indices between neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and that with other etiologies.
METHODS: Patients under 6 mo of age who were referred for investigation of conjugated hyperbilirubinaemia from June 2003 to December 2010 were eligible for this study. After excluding diseases affecting the extrahepatic biliary system, all patients were screened for the two most common SLC25A13 mutations; the coding exons of the entire SLC25A13 gene was sequenced and Western blotting of citrin protein performed in selected cases. Patients in whom homozygous or compound heterozygous SLC25A13 mutation and/or absence of normal citrin protein was detected were defined as having NICCD. Cases in which no specific etiological factor could be ascertained after a comprehensive conjugated hyperbilirubinaemia work-up were defined as idiopathic neonatal cholestasis (INC). Thirty-two NICCD patients, 250 INC patients, and 39 infants with cholangiography-confirmed biliary atresia (BA) were enrolled. Laboratory values at their first visit were abstracted from medical files and compared.
RESULTS: Compared with BA and INC patients, the NICCD patients had significantly higher levels of total bile acid (TBA) [all measures are expressed as median (inter-quartile range): 178.0 (111.2-236.4) μmol/L in NICCD vs 112.0 (84.9-153.9) μmol/L in BA and 103.0 (70.9-135.3) μmol/L in INC, P = 0.0001]. The NICCD patients had significantly lower direct bilirubin [D-Bil 59.6 (43.1-90.9) μmol/L in NICCD vs 134.0 (115.9-151.2) μmol/L in BA and 87.3 (63.0-123.6) μmol/L in INC, P = 0.0001]; alanine aminotransferase [ALT 34.0 (23.0-55.0) U/L in NICCD vs 108.0 (62.0-199.0) U/L in BA and 84.5 (46.0-166.0) U/L in INC, P = 0.0001]; aspartate aminotransferase [AST 74.0 (53.5-150.0) U/L in NICCD vs 153.0 (115.0-239.0) U/L in BA and 130.5 (81.0-223.0) U/L in INC, P = 0.0006]; albumin [34.9 (30.7-38.2) g/L in NICCD vs 38.4 (36.3-42.2) g/L in BA and 39.9 (37.0-42.3) g/L in INC, P = 0.0001]; glucose [3.2 (2.0-4.4) mmol/L in NICCD vs 4.1 (3.4-5.1) mmol/L in BA and 4.0 (3.4-4.6) mmol/L in INC, P = 0.0014] and total cholesterol [TCH 3.33 (2.97-4.00) mmol/L in NICCD vs 4.57 (3.81-5.26) mmol/L in BA and 4.00 (3.24-4.74) mmol/L in INC, P = 0.0155] levels. The D-Bil to total bilirubin (T-Bil) ratio was significantly lower in NICCD patients [all measures are expressed as median (inter-quartile range): 0.54 (0.40-0.74)] than that in BA patients [0.77 (0.72-0.81), P = 0.001] and that in INC patients [0.74 (0.59-0.80), P = 0.0045]. A much higher AST/ALT ratio was found in NICCD patients [2.46 (1.95-3.63)] compared to BA patients [1.38 (0.94-1.97), P = 0.0001] and INC patients [1.48 (1.10-2.26), P = 0.0001]. NICCD patients had significantly higher TBA/D-Bil ratio [3.36 (1.98-4.43) vs 0.85 (0.72-1.09) in BA patients and 1.04 (0.92-1.14) in INC patients, P = 0.0001], and TBA/TCH ratio [60.7 (32.4-70.9) vs 24.7 (19.8-30.2) in BA patients and 24.2 (21.4-26.9) in INC patients, P = 0.0001] compared to the BA and INC groups.
CONCLUSION: NICCD has significantly different biochemical indices from BA or INC. TBA excretion in NICCD appeared to be more severely disturbed than that of bilirubin and cholesterol.
PMCID: PMC3482648  PMID: 23112554
Cholestasis; Biliary atresia; Infants; Idiopathic neonatal cholestasis; SLC25A13
6.  Neonatal intrahepatic cholestasis caused by citrin deficiency: prevalence and SLC25A13 mutations among thai infants 
BMC Gastroenterology  2012;12:141.
The most common causes of cholestatic jaundice are biliary atresia and idiopathic neonatal hepatitis (INH). Specific disorders underlying INH, such as various infectious and metabolic causes, including neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) especially, in East Asian populations are increasingly being identified. Since most NICCD infants recovered from liver disease by 1 year of age, they often are misdiagnosed with INH, leading to difficulty in determining the true prevalence of NICCD. Mutation(s) of human SLC25A13 gene encoding a mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), can lead to AGC2 deficiency, resulting in NICCD and an adult-onset fatal disease namely citrullinemia type II (CTLN2). To study the prevalence of NICCD and SLC25A13 mutations in Thai infants, and to compare manifestations of NICCD and non-NICCD, infants with idiopathic cholestatic jaundice or INH were enrolled. Clinical and biochemical data were reviewed. Urine organic acid and plasma amino acids profiles were analyzed. PCR-sequencing of all 18 exons of SLC25A13 and gap PCR for the mutations IVS16ins3kb and Ex16+74_IVS17-32del516 were performed. mRNA were analyzed in selected cases with possible splicing error.
Five out of 39 (12.8%) unrelated infants enrolled in the study were found to have NICCD, of which three had homozygous 851del4 (GTATdel) and two compound heterozygous 851del4/IVS16ins3kb and 851del4/1638ins23, respectively. Two missense mutations (p.M1? and p.R605Q) of unknown functional significance were identified. At the initial presentation, NICCD patients had higher levels of alkaline phosphatase (ALP) and alpha-fetoprotein (AFP) and lower level of alanine aminotransferase (ALT) than those in non-NICCD patients (p< 0.05). NICCD patients showed higher citrulline level and threonine/serine ratio than non-NICCD infants (p< 0.05). Fatty liver was found in 2 NICCD patients. Jaundice resolved in all NICCD and in 87.5% of non-NICCD infants at the median age of 9.5 and 4.0 months, respectively.
NICCD should be considered in infants with idiopathic cholestasis. The preliminary estimated prevalence of NICCD was calculated to be 1/48,228 with carrier rate of 1/110 among Thai infants. However, this number may be underestimated and required further analysis with mutation screening in larger control population to establish the true prevalence of NICCD and AGC2 deficiency.
PMCID: PMC3483206  PMID: 23067347
AGC2 deficiency; Cholestatic jaundice; Idiopathic neonatal hepatitis; Infantile cholestasis; NICCD; Prevalence
7.  Isolation of Arabidopsis ahg11, a weak ABA hypersensitive mutant defective in nad4 RNA editing  
Journal of Experimental Botany  2012;63(14):5301-5310.
The phytohormone abscisic acid (ABA) plays pivotal roles in the regulation of developmental and environmental responses in plants. Identification of cytoplasmic ABA receptors enabled the elucidation of the main ABA signalling pathway, connecting ABA perception to either nuclear events or the action of several transporters. However, the physiological functions of ABA in cellular processes largely remain unknown. To obtain greater insight into the ABA response, genetic screening was performed to isolate ABA-related mutants of Arabidopsis and several novel ABA-hypersensitive mutants were isolated. One of those mutants—ahg11—was characterized further. Map-based cloning showed that AHG11 encodes a PPR type protein, which has potential roles in RNA editing. An AHG11-GFP fusion protein indicated that AHG11 mainly localized to the mitochondria. Consistent with this observation, the nad4 transcript, which normally undergoes RNA editing, lacks a single RNA editing event conferring a conversion of an amino acid residue in ahg11 mutants. The geminating ahg11 seeds have higher levels of reactive-oxygen-species-responsive genes. Presumably, partial impairment of mitochondrial function caused by an amino acid conversion in one of the complex I components induces redox imbalance which, in turn, confers an abnormal response to the plant hormone.
PMCID: PMC3430999  PMID: 22821940
Arabidopsis, abscisic acid, mitochondria, pentatricopeptide repeat protein, RNA editing
8.  Clostridium perfringens Iota-Toxin b Induces Rapid Cell Necrosis▿ 
Infection and Immunity  2011;79(11):4353-4360.
Clostridium perfringens iota-toxin is a binary toxin composed of an enzyme component (Ia) and a binding component (Ib). Each component alone lacks toxic activity, but together they produce cytotoxic effects. We examined the cytotoxicity of iota-toxin Ib in eight cell lines. A431 and A549 cells were susceptible to Ib, but MDCK, Vero, CHO, Caco-2, HT-29, and DLD-1 cells were not. Ib bound and formed oligomers in the membranes of A431 and MDCK cells. However, Ib entered MDCK cells but not A431 cells, suggesting that uptake is essential for cellular survival. Ib also induced cell swelling and the rapid depletion of cellular ATP in A431 and A549 cells but not the insensitive cell lines. In A431 cells, Ib binds and oligomerizes mainly in nonlipid rafts in the membranes. Disruption of lipid rafts by methyl-β-cyclodextrin did not impair ATP depletion or cell death caused by Ib. Ib induced permeabilization by propidium iodide without DNA fragmentation in A431 cells. Ultrastructural studies revealed that A431 cells undergo necrosis after treatment with Ib. Ib caused a disruption of mitochondrial permeability and the release of cytochrome c. Staining with active-form-specific antibodies showed that the proapoptotic Bcl-2-family proteins Bax and Bak were activated and colocalized with mitochondria in Ib-treated A431 cells. We demonstrate that Ib by itself produces cytotoxic activity through necrosis.
PMCID: PMC3257925  PMID: 21911469
9.  Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation 
The glutamate–glutamine cycle faces a drain of glutamate by oxidation, which is balanced by the anaplerotic synthesis of glutamate and glutamine in astrocytes. De novo synthesis of glutamate by astrocytes requires an amino group whose origin is unknown. The deficiency in Aralar/AGC1, the main mitochondrial carrier for aspartate–glutamate expressed in brain, results in a drastic fall in brain glutamine production but a modest decrease in brain glutamate levels, which is not due to decreases in neuronal or synaptosomal glutamate content. In vivo 13C nuclear magnetic resonance labeling with 13C2acetate or (1-13C) glucose showed that the drop in brain glutamine is due to a failure in glial glutamate synthesis. Aralar deficiency induces a decrease in aspartate content, an increase in lactate production, and lactate-to-pyruvate ratio in cultured neurons but not in cultured astrocytes, indicating that Aralar is only functional in neurons. We find that aspartate, but not other amino acids, increases glutamate synthesis in both control and aralar-deficient astrocytes, mainly by serving as amino donor. These findings suggest the existence of a neuron-to-astrocyte aspartate transcellular pathway required for astrocyte glutamate synthesis and subsequent glutamine formation. This pathway may provide a mechanism to transfer neuronal-born redox equivalents to mitochondria in astrocytes.
PMCID: PMC3049464  PMID: 20736955
AGC1; Aralar; aspartate; glial glutamine; mitochondrial aspartate–glutamate carrier; OmniBank
10.  Identification and characterization of the RNA binding surface of the pentatricopeptide repeat protein 
Nucleic Acids Research  2011;40(6):2712-2723.
The expressions of chloroplast and mitochondria genes are tightly controlled by numerous nuclear-encoded proteins, mainly at the post-transcriptional level. Recent analyses have identified a large, plant-specific family of pentatricopeptide repeat (PPR) motif-containing proteins that are exclusively involved in RNA metabolism of organelle genes via sequence-specific RNA binding. A tandem array of PPR motifs within the protein is believed to facilitate the RNA interaction, although little is known of the mechanism. Here, we describe the RNA interacting framework of a PPR protein, Arabidopsis HCF152. First, we demonstrated that a Pfam model could be relevant to the PPR motif function. A series of proteins with two PPR motifs showed significant differences in their RNA binding affinities, indicating functional differences among PPR motifs. Mutagenesis and informatics analysis putatively identified five amino acids organizing its RNA binding surface [the 1st, 4th, 8th, 12th and ‘ii’(-2nd) amino acids] and their complex connections. SELEX (Systematic evolution of ligands by exponential enrichment) and nucleobase preference assays determined the nucleobases with high affinity for HCF152 and suggested several characteristic amino acids that may be involved in determining specificity and/or affinity of the PPR/RNA interaction.
PMCID: PMC3315335  PMID: 22127869
11.  Clostridium perfringens TpeL Glycosylates the Rac and Ras Subfamily Proteins▿  
Infection and Immunity  2010;79(2):905-910.
Clostridium perfringens TpeL belongs to a family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A (TcdA) and B (TcdB) and Clostridium sordellii lethal toxin (TcsL). We report here the identification of the TpeL-catalyzed modification of small GTPases. A recombinant protein (TpeL1-525) derived from the TpeL N-terminal catalytic domain in the presence of streptolysin O (SLO) induced the rounding of Vero cells and the glycosylation of cellular Rac1. Among several hexoses tested, UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-glucose (UDP-Glc) served as cosubstrates for TpeL1-525-catalyzed modifications. TpeL1-525 catalyzed the incorporation of UDP-Glc into Ha-Ras, Rap1B, and RalA and of UDP-GlcNAc into Rac1, Ha-Ras, Rap1B, and RalA. In Rac1, TpeL and TcdB share the same acceptor amino acid for glycosylation, Thr-35. In Vero cells treated with TpeL1-525 in the presence of SLO, glycosylation leads to a translocation of the majority of Rac1 and Ha-Ras to the membrane. We demonstrate for first time that TpeL uses both UDP-GlcNAc and UDP-Glc as donor cosubstrates and modifies the Rac1 and Ras subfamily by glycosylation to mediate its cytotoxic effects.
PMCID: PMC3028844  PMID: 21098103
12.  Etiological Analysis of Neurodevelopmental Disabilities: Single-Center Eight-Year Clinical Experience in South China 
Etiology determination of neurodevelopmental disabilities (NDDs) currently remains a worldwide common challenge on child health. We herein reported the etiology distribution feature in a cohort of 285 Chinese patients with NDDs. Although concrete NDD etiologies in 48.4% of the total patients could not be identified, genetic diseases (with the proportion of 35.8% in the total cases) including inborn errors of metabolism (IEM) and congenital dysmorphic diseases, constituted the commonest etiology category for NDDs in this study. The two key experimental technologies in pediatric metabolomics, gas chromatography-mass spectrometry (GC-MS), and tandem mass spectrometry (MS-MS), proved to be substantially helpful for the exploration of the NDD etiologies in this clinical investigation. The findings in this paper provided latest epidemiologic information on the etiology distribution of NDDs in Chinese, and the syndromic NDDs caused by citrin deficiency and the novel chromosomal karyotype, respectively, further expanded the etiology spectrum of NDDs.
PMCID: PMC2948914  PMID: 20936111
13.  Clostridium perfringens Iota-Toxin: Structure and Function 
Toxins  2009;1(2):208-228.
Clostridium perfringens iota-toxin is composed of the enzyme component (Ia) and the binding component (Ib). Ib binds to receptor on targeted cells and translocates Ia into the cytosol of the cells. Ia ADP-ribosylates actin, resulting in cell rounding and death. Comparisons of the deduced amino acid sequence from the gene and three-dimensional structure of Ia with those of ADP-ribosylating toxins (ARTs) suggests that there is striking structural similarity among these toxins. Our objectives are to review the recent advances in the character, structure-function, and the mode of action of iota-toxin by consideration of the findings about ARTs.
PMCID: PMC3202787  PMID: 22069542
Clostridium perfringens; iota-toxin; ADP-ribosylating toxin; NAD+-glycohydrolase; ADP-ribosyltransferase; crystal structure; SN1 mechanism; internalization; endocytosis
14.  Complete blockage of the mevalonate pathway results in male gametophyte lethality 
Journal of Experimental Botany  2009;60(7):2055-2064.
Plants have two isoprenoid biosynthetic pathways: the cytosolic mevalonate (MVA) pathway and the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Since the discovery of the MEP pathway, possible metabolic cross-talk between these pathways has prompted intense research. Although many studies have shown the existence of such cross-talk using feeding experiments, it remains to be determined if native cross-talk, rather than exogenously applied metabolites, can compensate for complete blockage of the MVA pathway. Previously, Arabidopsis mutants for HMG1 and HMG2 encoding HMG-CoA reductase (HMGR) were isolated. Although it was shown that HMGR1 is a functional HMGR, the enzyme activity of HMGR2 has not been confirmed. It is demonstrated here that HMG2 encodes a functional reductase with similar activity to HMGR1, using enzyme assays and complementation experiments. To estimate the contribution of native cross-talk, an attempt was made to block the MVA pathway by making double mutants lacking both HMG1 and HMG2, but no double homozygotes were detected in the progeny of self-pollinated HMG1/hmg1 hmg2/hmg2 plants. hmg1 hmg2 male gametophytes appeared to be lethal based on crossing experiments, and microscopy indicated that ∼50% of the microspores from the HMG1/hmg1 hmg2/hmg2 plant appeared shrunken and exhibited poorly defined endoplasmic reticulum membranes. In situ hybridization showed that HMG1 transcripts were expressed in both the tapetum and microspores, while HMG2 mRNA appeared only in microspores. It is concluded that native cross-talk from the plastid cannot compensate for complete blockage of the MVA pathway, at least during male gametophyte development, because either HMG1 or HMG2 is required for male gametophyte development.
PMCID: PMC2682496  PMID: 19363204
Anther; cross-talk; HMG-CoA reductase; isoprenoid; male gametophyte; MEP pathway; MVA pathway; pollen; sterol; tapetum
15.  An Alternative Approach to the Monitoring of Respiration by Dynamic Air-Pressure Sensor 
Anesthesia Progress  2007;54(1):2-6.
Monitoring and assessing of patient respiratory function during conscious sedation are important because many drugs used for conscious sedation produce respiratory depression and subsequent hypoventilation. The purpose of this study is to assess the value of a dynamic air-pressure sensor for respiratory monitoring of clothed patients. Eight clothed adult volunteers were reclined on a dental chair positioned horizontally. The air bag for measuring air-pressure signals corresponding to respiration was placed on the seat back of the dental chair in the central lumbar area of the subject. The subject breathed through a face mask with a respirometer attached for measuring expiratory tidal volume. The air-pressure signals corresponding to respiration were obtained and the time integration values for air pressure during each expiration (∫Pexp) were calculated. The expiratory tidal volume (TVexp) was measured simultaneously by respirometer. The relationship between TVexp and ∫Pexp for each subject was assessed by a Pearson correlation coefficient. A strong correlation between TVexp and ∫Pexp was observed in all subjects. Measuring ∫Pexp by dynamic air-pressure sensor makes it possible to estimate respiratory volume breath by breath, and the respiratory pressure–time integral waveform was useful in visually monitoring the respiration pattern. We believe that in the future this device will be used to monitor respiratory physiology in clothed patients, contributing to safer sedative procedures.
PMCID: PMC1821134  PMID: 17352526
Air-pressure sensor; Respiratory; Monitor; Nonrestrictively
16.  Identification of the Imprinted KLF14 Transcription Factor Undergoing Human-Specific Accelerated Evolution  
PLoS Genetics  2007;3(5):e65.
Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the gene's expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage.
Author Summary
Imprinted genes are expressed in a parent-of-origin manner, where one of the two inherited copies of the imprinted gene is silenced. Aberrations in the expression of these genes, which generally regulate growth, are associated with various developmental disorders, emphasizing the importance of their discovery and analysis. In this study, we identify a novel imprinted gene, named KLF14, on human Chromosome 7. It is predicted to bind DNA and regulate transcription and was shown to be expressed from the maternally inherited chromosome in all human and mouse tissues examined. Surprisingly, we did not identify molecular signatures generally associated with imprinted regions, such as DNA methylation. Additionally, the identification of numerous DNA sequence variants led to an in-depth analysis of the gene's evolution. It was determined that there is greater variability in KLF14 in the human lineage, when compared to other primates, with a significantly increased number of polymorphisms encoding for changes at the protein level, suggesting human-specific accelerated evolution. As the first example of an imprinted transcript undergoing accelerated evolution in the human lineage, we propose that the accumulation of polymorphisms in KLF14 may be aided by the silencing of the inactive allele, allowing for stronger selection.
PMCID: PMC1865561  PMID: 17480121
17.  Binding and Internalization of Clostridium perfringens Iota-Toxin in Lipid Rafts  
Infection and Immunity  2004;72(6):3267-3275.
Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). The oligomer of Ib formed in membranes induces endocytosis. We examined the binding and internalization of Ib by using Cy3-labeled Ib. Labeled Ib was retained at the membranes of MDCK cells for 60 min of incubation at 37°C, and later it was detected in cytoplasmic vesicles. To determine whether Ib associates with lipid rafts, we incubated MDCK cells with Ib at 4 or 37°C and fractionated the Triton-insoluble membranes. An Ib complex of 500 kDa was localized at 37°C to the insoluble fractions that fulfilled the criteria of lipid rafts, but it did not form at 4°C. The amount of complex in the raft fraction reached a maximum after 60 min of incubation at 37°C. When the cells that were preincubated with Ib at 4°C were incubated at 37°C, the complex was detected in the raft fraction. The treatment of MDCK cells with methyl-β-cyclodextrin reduced the localization of the Ib complex to the rafts and the rounding of the cells induced by Ia plus Ib. When 125I-labeled Ia was incubated with the cells in the presence of Ib at 37°C, it was localized in the raft fraction. Surface plasmon resonance analysis revealed that Ia binds to the oligomer of Ib. We conclude that Ib binds to a receptor in membranes and then moves to rafts and that Ia bound to the oligomer of Ib formed in the rafts is internalized.
PMCID: PMC415663  PMID: 15155629
18.  Slc25a13-Knockout Mice Harbor Metabolic Deficits but Fail To Display Hallmarks of Adult-Onset Type II Citrullinemia 
Molecular and Cellular Biology  2004;24(2):527-536.
Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn−/− mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn−/− mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.
PMCID: PMC343808  PMID: 14701727
19.  Binding Component of Clostridium perfringens Iota-Toxin Induces Endocytosis in Vero Cells  
Infection and Immunity  2002;70(4):1909-1914.
Clostridium perfringens iota-toxin is a binary toxin consisting of two individual proteins, the binding component (Ib) and the enzyme component (Ia). Wild-type Ib bound to Vero cells at 4 and 37°C and formed oligomers at 37°C but not at 4°C. The Ib-induced K+ release from the cells was dependent on the oligomer formation of Ib in the cells, but the oligomer formation did not induce rounding activity or cytotoxicity. After incubation of the cells with recombinant Ib (rIb) at 37°C, the Ib oligomer in the cell became resistant to pronase treatment with time, but the Ib monomer was sensitive to the treatment. Furthermore, treatment of Vero cells with rIb in the presence of bafilomycin, methylamine, or ethylamine resulted in accumulation of the oligomer in the cells but had no effect on K+ release. Moreover, incubation with Ib plus Ia in the presence of these agents caused no rounding in the cells. These observations suggest that Ib binds to Vero cells, itself oligomerizing to form ion-permeable channels and that the formation of oligomer then induces endocytosis.
PMCID: PMC127877  PMID: 11895954
20.  Characterization of the Enzymatic Component of Clostridium perfringens Iota-Toxin 
Journal of Bacteriology  2000;182(8):2096-2103.
The iotaa component (ia) of Clostridium perfringens ADP ribosylates nonmuscle β/γ actin and skeletal muscle α-actin. Replacement of Arg-295 in ia with alanine led to a complete loss of NAD+-glycohydrolase (NADase) and ADP-ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity (<0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD+ to ia, that Arg-295, Glu-380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in ia on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in ia are dependent on ARTase activity.
PMCID: PMC111256  PMID: 10735850

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