Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense

Background: Since Trypanosorna spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life -cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T brucei Hp-Hb complex receptor (TbHpHbR). Methods: Trypanosorna congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay. Results: We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K,A) =2.1x10(-8) M) but free-Hp with low affinity (Kd = 2.2x10(-7) M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome). Conclusion: We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T congolense EMF-specific Hb receptor (TcEpHbR)

Takotsubo cardiomyopathy with marked ST-segment elevation and electrical alternans complicated with hyperglycemic hyperosmolar state

金沢大学医薬保健研究域医学系This is the first report of a case of Takotsubo cardiomyopathy with a hyperglycemic hyperosmolar state (HHS). This case presented with marked ST-segment elevation and electrical alternans, uncommon findings in Takotsubo cardiomyopathy. We believe that hyperosmolarity-induced myocardial dehydration and consequent increase in intracellular calcium concentration may be the mechanism of Takotsubo cardiomyopathy and electrical alternans in HH

Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases

Aims The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them. Methods and Results We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analyzed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as “pathogenic” by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that 2 variants in KCNH2 and SCN5A, 4 variants in SCN10A, and 1 variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from “Uncertain significance” to “Likely pathogenic” in 6 probands. Conclusions Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD. Translational Perspective Whole-exome sequencing (WES) may be helpful in determining the causes of cardiac conduction system disease (CCSD), however, the identification of pathogenic variants remains a challenge. We performed WES of 23 probands diagnosed with early-onset CCSD, and identified 12 pathogenic or likely pathogenic variants in 11 of these probands (48%) according to the 2015 ACMG standards and guidelines. In this context, functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants, and SCN10A may be one of the major development factors in CCSD

The Diamond STING server

Diamond STING is a new version of the STING suite of programs for a comprehensive analysis of a relationship between protein sequence, structure, function and stability. We have added a number of new functionalities by both providing more structure parameters to the STING Database and by improving/expanding the interface for enhanced data handling. The integration among the STING components has also been improved. A new key feature is the ability of the STING server to handle local files containing protein structures (either modeled or not yet deposited to the Protein Data Bank) so that they can be used by the principal STING components: (Java)Protein Dossier ((J)PD) and STING Report. The current capabilities of the new STING version and a couple of biologically relevant applications are described here. We have provided an example where Diamond STING identifies the active site amino acids and folding essential amino acids (both previously determined by experiments) by filtering out all but those residues by selecting the numerical values/ranges for a set of corresponding parameters. This is the fundamental step toward a more interesting endeavor—the prediction of such residues. Diamond STING is freely accessible at and

Multiple noncoding exons 1 of nuclear receptors NR4A family (nerve growth factor-induced clone B, Nur-related factor 1 and neuron-derived orphan receptor 1) and NR5A1 (steroidogenic factor 1) in human cardiovascular and adrenal tissues

金沢大学医薬保健研究域医学系Objective: Nuclear receptors are involved in a wide variety of functions, including aldosteronogenesis. Nuclear receptor families NR4A [nerve growth factor-induced clone B (NGFIB), Nur-related factor 1 (NURR1) and neuron-derived orphan receptor 1 (NOR1)] and NR2F [chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TFI), COUP-TFII and NR2F6) activate, whereas NR5A1 [steroidogenic factor 1 (SF1)] represses CYP11B2 (aldosterone synthase) gene transcription. The present study was undertaken to elucidate the mechanism of differential regulation of nuclear receptors between cardiovascular and adrenal tissues. Methods: We collected tissues of artery (n = 9), cardiomyopathy muscle (n = 9), heart muscle (noncardiomyopathy) (n = 6), adrenal gland (n = 9) and aldosterone-producing adenoma (APA) (n = 9). 5′-rapid amplification of cDNA ends (RACE) identified transcription start sites. Multiplex reverse-transcription PCR (RT-PCR) determined use of alternative noncoding exons 1 (ANEs). Results: In adrenocortical H295R cells, angiotensin II, KCl or cAMP, all stimulated CYP11B2 transcription and NR4A was upregulated, whereas NR2F and NR5A1 were downregulated. 5′-RACE and RT-PCR revealed four ANEs of NGFIB (NR4A1), three of NURR1 (NR4A2), two of NOR1 (NR4A3) and two of SF1 (NR5A1) in cardiovascular and adrenal tissues. Quantitative multiplex RT-PCR showed NR4A and NR5A1 differentially employed multiple ANEs in a tissue-specific manner. The use of ANEs of NGFIB and NURR1 was significantly different between APA and artery. Changes in use of ANEs of NGFIB and NOR1 were observed between cardiomyopathy and noncardiomyopathy. The NR4A mRNA levels in artery were high compared with cardiac and adrenal tissues, whereas the NR5A1 mRNA level in adrenal tissues was extremely high compared with cardiovascular tissues. Conclusion: NR4A and NR5A1 genes are complex in terms of alternative promoter use. The use of ANEs may be associated with the pathophysiology of the heart and adrenal gland. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

High sensitivity of late gadolinium enhancement for predicting microscopic myocardial scarring in biopsied specimens in hypertrophic cardiomyopathy

金沢大学医薬保健研究域医学系Background: Myocardial scarring can be assessed by cardiac magnetic resonance imaging with late gadolinium enhancement and by endomyocardial biopsy. However, accuracy of late gadolinium enhancement for predicting microscopic myocardial scarring in biopsied specimens remains unknown in hypertrophic cardiomyopathy. We investigated whether late gadolinium enhancement in the whole heart reflects microscopic myocardial scarring in the small biopsied specimens in hypertrophic cardiomyopathy. Methods and Results: Twenty-one consecutive patients with hypertrophic cardiomyopathy who were examined both by cardiac magnetic resonance imaging and by endomyocardial biopsy were retrospectively studied. The right interventricular septum was the target site for endomyocardial biopsy in all patients. Late gadolinium enhancement in the ventricular septum had an excellent sensitivity (100%) with a low specificity (40%) for predicting microscopic myocardial scarring in biopsied specimens. The sensitivity of late gadolinium enhancement in the whole heart remained 100% with a specificity of 27% for predicting microscopic myocardial scarring in biopsied specimens. Quantitative assessments of fibrosis revealed that the extent of late gadolinium enhancement in the whole heart was the only independent variable related to the microscopic collagen fraction in biopsied specimens (β = 0.59, 95% confident interval: 0.15-1.0, p = 0.012). Conclusions: Although there was a compromise in the specificity, the sensitivity of late gadolinium enhancement was excellent for prediction of microscopic myocardial scarring in hypertrophic cardiomyopathy. Moreover, the severity of late gadolinium enhancement was independently associated with the quantitative collagen fraction in biopsied specimens in hypertrophic cardiomyopathy. These findings indicate that late gadolinium enhancement can reflect both the presence and the extent of microscopic myocardial scarring in the small biopsied specimens in hypertrophic cardiomyopathy. © 2014 Konno et al