The effect of metal ions on the Spf1p P5A-ATPase. High sensitivity to irreversible inhibition by zinc.

The Spf1p protein from Saccharomyces cerevisiae belongs to the family of P5A-ATPases that have recently been shown to protect the endoplasmic reticulum by dislocating misinserted membrane proteins. The loss of function of P5A-ATPases leads to endoplasmic reticulum stress with a pleiotropic phenotype including protein, sterol and metal ion dyshomeostasis. Like other P-ATPases, Spf1p requires Mg2+. We found that free Mg2+ stimulated the Spf1p ATPase activity along a double hyperbolic curve with two components of K1/2 = 14 and 800 µM Ca2+, Mn2+ and Co2+ lowered about 50% of the Spf1p ATPase with relatively low affinity (Ki ∼75 µM) and the activity was fully recovered after metal ion chelation with EGTA. In contrast, low concentrations of Zn2+ and Cd2+decreased the activity to less than 20% and lead to slow irreversible inactivation of the enzyme. After the treatment with Zn2+, Spf1p exhibited a reduced apparent affinity for ATP and formed lower levels of the catalytic phosphoenzyme. The inactivation by Zn2+ occurred preferentially at a pH > 6 and could be prevented by adding either ATP or ADP to the inactivation media. These results suggest that Zn2+ inactivated Spf1p by binding to amino acid residues from the nucleotide binding-phosphorylation domains that are protonated at lower pH. Alternatively the binding of nucleotides may indirectly compete with a conformational change leading to the Zn2+-inactive form of the enzyme. Exposure of yeast cells to high concentrations of Zn2+ led to changes similar to the phenotype characteristic of the Spf1Δ cells. Altogether, our data, point out a possible mechanism by which the inhibition of P5A-ATPases could potentiate metal ion-induced ER stress and proteotoxicity.

The Spf1p P5A-ATPase "arm-like" domain is not essential for ATP hydrolysis but its deletion impairs autophosphorylation.

The yeast Spf1p P5A-ATPase actively translocates membrane spanning peptides of mislocalized proteins from the endoplasmic reticulum. Loss of Spf1p function causes a pleiotropic ER stress-phenotype associated with alterations of homeostasis of metal ions, lipids, protein folding, glycosylation, and membrane insertion. A unique characteristic of P5A-ATPases is the presence of an extended insertion which was called the "arm-like" domain connecting the phosphorylation domain (P) with transmembrane segment M5 near the peptidyl-substrate binding pocket. Here we have constructed and characterized a Δarm mutant of Spf1p lacking a segment of 117 amino acids of the "arm-like" domain. The Δarm mutant was capable of hydrolyzing ATP at maximal rates of 50% of that of the wild type enzyme. With the non-nucleotide substrate analog pNPP, the hydrolytic activity of the mutant dropped to 10%. The mutant showed an apparent affinity for ATP similar to the wild type. When incubated with ATP the Δarm mutant produced a lower level of the catalytic phosphoenzyme in amounts proportionate to the ATPase activity. These results indicate that the "arm-like" domain is not essential for hydrolytic activity and suggest that it is needed for the stabilization of Spf1p in a phosphorylation-ready conformation.

Leucoencefalopatía posterior reversible en pacientes trasplantados. Presentación clínica, radiológica y evolución / Posterior reversible encephalopathy syndrome in transplanted patients. Clinical features and imaging follow up

Resumen El síndrome de leucoencefalopatía posterior reversible (PRES) es un desorden neurológico agudo caracterizado por cefalea, alteración de la conciencia, convulsiones y alteraciones visuales, con imágenes de edema vasogénico reversible en regiones cerebrales posteriores. Nos propusimos describir una serie de casos de pacientes trasplantados que desarrollaron PRES, caracterizando su presentación, evolución clínica, imágenes y terapéutica. Se analizaron historias clínicas informatizadas desde enero 2009 hasta enero 2019. Se recabaron datos demográficos, antecedentes clínicos, motivos y días de internación, tiempos desde el trasplante a la presentación clínica y diagnóstico. Se evaluó la mejoría/resolución en estudios por imágenes y la supervivencia anual. Se identificaron 27 pacientes con PRES; 22 trasplantados de órgano sólido de 1647 totales (1.3%) y 5 de médula ósea de 617 totales (0.8%). La media de edad fue de 38.2 años (DS 19.5), 62.9% de sexo femenino, 59.2% (16) antes del año del trasplante. Las comorbilidades más frecuentes enfermedad renal (14; 51%) e hipertensión arterial (11; 40%). Se realizó tomografía axial computarizada (TAC) a 23 pacientes (85.1%), siendo patológica en 11 (47.8%), y resonancia magnética nuclear (RMN) a 25 (92.6%), mostrando patrón característico en 17 (62.9%), con mejoría/resolución antes del año en 20 (74%). El tratamiento fue sintomático, modificando la inmunosupresión. Se registraron 5 óbitos durante la internación y otros 3 antes del año, con una supervivencia anual del 70.3% (19). La población de trasplantados, en crecimiento en nuestro medio, es particularmente susceptible al PRES. Tanto su presentación en estudios por imágenes, como su comorbilidad, difieren de otras poblaciones.

Abstract Posterior reversible encephalopathy syndrome (PRES) is an acute neurological disorder characterized by headache, encephalopathy, seizures and visual disturbances, with reversible vasogenic edema in posterior brain areas. The aim of this research was to describe a case series of transplanted patients who developed PRES, characterize their presentation, treatment, clinical and imaging evolution. Electronic medi cal records were analyzed from January 2009 to January 2019. Demographic data, clinical backgrounds, causes of admission, hospital length of stay and time from transplantation to PRES were collected. Image improvement/ resolution and annual survival were assessed. We identified 27 patients with PRES; 22 of 1647 total solid-organ transplant (1.3%) and 5 of 617 total bone marrow transplant (0.8%). The mean age at presentation was 38.2 years (SD 19.5), 62.9% female, 59.2% (16) before the year of transplantation. The most common comorbidities were kidney disease (14; 51%) and high blood pressure (11; 40%). Computed axial tomography (CT) was per formed in 23 patients (85.1%), with pathological findings in 11 (47.8%). Magnetic resonance imaging (MRI) of 25 (92.6%), showed a characteristic pattern in 17 (62.9%) with improvement/resolution before the year in 20 (74%). Treatment was symptomatic, modifying immunosuppression. Five deaths were recorded during hospital stay and another 3 before the year of admission, with an annual survival of 70.3% (19 patients). Organ transplant trend is growing in our region. These patients are particularly susceptible to PRES, with a different imaging presentation and comorbidities from other populations.

Replacing the Z-phenyl Ring in Tamoxifen® with a para-Connected NCN Pincer-Pt-Cl Grouping by Post-Modification †.

Post-modification of a series of NCN-pincer platinum(II) complexes [PtX(NCN-R-4)] (NCN = [C6H2(CH2NMe2)2-2,6]-, R = C(O)H, C(O)Me and C(O)Et), X = Cl- or Br-) at the para-position using the McMurry reaction was studied. The synthetic route towards two new [PtCl(NCN-R-4)] (R = C(O)Me and C(O)Et) complexes used above is likewise described. The utility and limitations of the McMurry reaction involving these pincer complexes was systematically evaluated. The predicted "homo-coupling" reaction of [PtBr(NCN-C(O)H-4)] led to the unexpected formation of 3,3',5,5'-tetra[(dimethylamino)methyl]-4,4'-bis(platinum halide)-benzophenone (halide = Br or Cl), referred to hereafter as the bispincer-benzophenone complex 13. This material was further characterized using X-ray crystal structure determination. The applicability of the pincer complexes in the McMurry reaction is shown to open a route towards the synthesis of tamoxifen-type derivatives of which one phenyl ring of Tamoxifen® itself is replaced by an NCN arylplatinum pincer fragment. The newly synthesized derivatives can be used as potential candidates in anti-cancer drug screening protocols. Two NCN-arylpincer platinum tamoxifen type derivatives, 5 and 6, were successfully synthesized and of 5 the separation of the diastereomeric E-/Z-forms was achieved. Compound 6, which is the pivaloyl protected NCN pincer platinum hydroxy-Tamoxifen® derivative, was obtained as a mixture of E-/Z-isomers. The new derivatives were further analyzed and characterized with 1H-, 13C{1H}- and 195Pt{1H}-NMR, IR, exact mass MS and elemental analysis.

Reduction of the P5A-ATPase Spf1p phosphoenzyme by a Ca2+-dependent phosphatase.

P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D487N) also exhibited Ca2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a co-purifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca2+ or Zn2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca2+. These results indicate that the reported effects of Ca2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase.

Multidimensional Evaluation of Endogenous and Health Factors Affecting Food Preferences, Taste and Smell Perception.

OBJECTIVE: This study, by taking a holistic approach, investigates the relationships between taste, smell sensitivity and food preference with prognostic (endogenous and health) factors including age, gender, genetic taste markers, body mass, cigarette smoking, and number of drugs used. DESIGN: Cross sectional study. SETTING: Northern Italy. PARTICIPANTS: 203 healthy subjects (160 women/43 men; mean age: 58.2±19.8 years) were examined. MEASUREMENTS: Individual taste sensitivity was determined by saccharose, sodium chloride, acetic acid and caffeine solutions and by 6-n-propylthiouracil (PROP) responsiveness test. Olfactory sensitivity has been assessed by «Sniffin' Sticks¼. Four tag Single nucleotide polymorphisms (SNPs) in regions of interest were genotyped. Factor analysis and multivariate regression were performed for scaling food preferences and screening prognostic factors, respectively. RESULTS: Increasing age is associated with decreased responsiveness to NaCl (P=0.001), sweet solutions (P=0.044), and smell perception (P<0.001). Concerning the food preferences, elderly like the "vegetables" and "fruits" but dislike "spicy" more than younger. Regarding number of drugs taken, there is a significant negative effect on smell perception (P<0.001). In addition, drugs reduce both the "vegetables foods" score (P=0.002) and the "milk-product foods" score (P=0.027). With respect to Body Mass Index (BMI), only a significant effect was shown, on sweet perception (P=0.006). Variation in taste receptor genes can give rise to differential perception of sweet, acid and bitter tastes. No effect of gender and smoking was observed. CONCLUSIONS: Our study suggested that age, genetic markers, BMI and drugs use are the factors which affect taste and smell perception and food preferences.

Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing.

PURPOSE: Germ-line testing for panels of cancer genes using next-generation sequencing is becoming more common in clinical care. We report our experience as a clinical laboratory testing both well-established, high-risk cancer genes (e.g., BRCA1/2, MLH1, MSH2) as well as more recently identified cancer genes (e.g., PALB2, BRIP1), many of which have increased but less well-defined penetrance. METHODS: Clinical genetic testing was performed on over 10,000 consecutive cases referred for evaluation of germ-line cancer genes, and results were analyzed for frequency of pathogenic or likely pathogenic variants, and were stratified by testing panel, gene, and clinical history. RESULTS: Overall, a molecular diagnosis was made in 9.0% of patients tested, with the highest yield in the Lynch syndrome/colorectal cancer panel. In patients with breast, ovarian, or colon/stomach cancer, positive yields were 9.7, 13.4, and 14.8%, respectively. Approximately half of the pathogenic variants identified in patients with breast or ovarian cancer were in genes other than BRCA1/2. CONCLUSION: The high frequency of positive results in a wide range of cancer genes, including those of high penetrance and with clinical care guidelines, underscores both the genetic heterogeneity of hereditary cancer and the usefulness of multigene panels over genetic tests of one or two genes.Genet Med 18 8, 823-832.

White phosphorus degradation with a NacNac aluminum carbene analogue: the biradical reaction mechanism.

The title compound Al-NacNac is isolobal to the imidazol-2-ylidene (NHC); the latter is considered as a nucleophilic carbene. However, the title compound is different from a typical carbene, as aluminum is a heavier group 13 element with a predominant inert s orbital. Its singlet ground state is a poor Lewis donor (acceptor) toward white phosphorus, but its corresponding lowest energy triplet state forms a strong Al-P bond with (opened) white phosphorus. The reaction of Al-NacNac with white phosphorus proceeds in two steps: after the addition of a first carbene analogue, a second one is added, resulting in a transient biradicaloid species. This undergoes facile subsequent rearrangement, and a final ring closure reaction leads to the observed product with a bicyclobutane moiety. It is determined by intramolecular bond formation of two phosphorus centered radicals. Finally, a structure with a large singlet-triplet energy separation is formed. An analogy to the noninnocent ligand character as well as the exciplex view of the monoadduct of white phosphorus with the Al-NacNac system is drawn.

Microfluidic titer plate for stratified 3D cell culture.

Human tissues and organs are inherently heterogeneous. Their functionality is determined by the interplay between different cell types, their secondary architecture, vascular system and gradients of signaling molecules and metabolites. Here we propose a stratified 3D cell culture platform, in which adjacent lanes of gels and liquids are patterned by phaseguides to capture this tissue heterogeneity. We demonstrate 3D cell culture of HepG2 hepatocytes under continuous perfusion, a rifampicin toxicity assay and co-culture with fibroblasts. 4T1 breast cancer cells are used to demonstrate invasion and aggregation models. The platform is incorporated in a microtiter plate format that renders it fully compatible with automation and high-content screening equipment. The extended functionality, ease of handling and full compatibility to standard equipment is an important step towards adoption of Organ-on-a-Chip technology for screening in an industrial setting.

Torsades de pointes following acute myocardial infarction: evidence for a deadly link with a common genetic variant.

BACKGROUND: Although QT prolongation following myocardial infarction (MI) is generally moderate, cases with marked QT prolongation leading to life-threatening torsades de pointes (TdP) have been described. OBJECTIVE: To investigate the genetic substrate of this phenomenon. METHODS: We studied 13 patients who developed TdP in the subacute phase of MI (2-11 days) and a group of 133 ethnically matched controls with uncomplicated MI. Long QT syndrome genes and the KCNH2-K897T polymorphism were screened by using denaturing high-performance liquid chromatography plus direct sequencing and a specific TaqMan assay, respectively. RESULTS: Two of the 13 patients (15%) who presented with QT prolongation and TdP were found to carry long QT syndrome mutations (KCNH2-R744X and SCN5A-E446K). Nine of the remaining 11 patients (82%) carried the KCNH2-K897T polymorphism, which was present in 35% of the controls (P = .0035). Thus, patients with an acute MI carrying the KCNH2-K897T polymorphism had an 8-fold greater risk of experiencing TdP compared with controls (95% confidence interval = 2-40). CONCLUSIONS: Our data suggest that the common K897T polymorphism is associated with an increased risk of TdP developing in the subacute phase of MI. Our findings support the concept that the electrical remodeling associated with this healing phase of MI may unmask a genetic substrate predisposing to a time-limited development of life-threatening arrhythmias. They also provide the first line of evidence in support of the hypothesis that a common polymorphism, previously described as a modifier of the severity of LQTS, may increase the risk of life-threatening arrhythmias in a much more prevalent cardiac disease such as myocardial infarction.

Distinguishing arrhythmogenic right ventricular cardiomyopathy/dysplasia-associated mutations from background genetic noise.

OBJECTIVES: The aims of this study were to determine the spectrum and prevalence of "background genetic noise" in the arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC) genetic test and to determine genetic associations that can guide the interpretation of a positive test result. BACKGROUND: ARVC is a potentially lethal genetic cardiovascular disorder characterized by myocyte loss and fibrofatty tissue replacement of the right ventricle. Genetic variation among the ARVC susceptibility genes has not been systematically examined, and little is known about the background noise associated with the ARVC genetic test. METHODS: Using direct deoxyribonucleic acid sequencing, the coding exons/splice junctions of PKP2, DSP, DSG2, DSC2, and TMEM43 were genotyped for 93 probands diagnosed with ARVC from the Netherlands and 427 ostensibly healthy controls of various ethnicities. Eighty-two additional ARVC cases were obtained from published reports, and additional mutations were included from the ARVD/C Genetic Variants Database. RESULTS: The overall yield of mutations among ARVC cases was 58% versus 16% in controls. Radical mutations were hosted by 0.5% of control individuals versus 43% of ARVC cases, while 16% of controls hosted missense mutations versus a similar 21% of ARVC cases. Relative to controls, mutations in cases occurred more frequently in non-Caucasians, localized to the N-terminal regions of DSP and DSG2, and localized to highly conserved residues within PKP2 and DSG2. CONCLUSIONS: This study is the first to comprehensively evaluate genetic variation in healthy controls for the ARVC susceptibility genes. Radical mutations are high-probability ARVC-associated mutations, whereas rare missense mutations should be interpreted in the context of race and ethnicity, mutation location, and sequence conservation.

Spectrum and prevalence of mutations from the first 2,500 consecutive unrelated patients referred for the FAMILION long QT syndrome genetic test.

BACKGROUND: Long QT syndrome (LQTS) is a potentially lethal, highly treatable cardiac channelopathy for which genetic testing has matured from discovery to translation and now clinical implementation. OBJECTIVES: Here we examine the spectrum and prevalence of mutations found in the first 2,500 unrelated cases referred for the FAMILION LQTS clinical genetic test. METHODS: Retrospective analysis of the first 2,500 cases (1,515 female patients, average age at testing 23 +/- 17 years, range 0 to 90 years) scanned for mutations in 5 of the LQTS-susceptibility genes: KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6). RESULTS: Overall, 903 referral cases (36%) hosted a possible LQTS-causing mutation that was absent in >2,600 reference alleles; 821 (91%) of the mutation-positive cases had single genotypes, whereas the remaining 82 patients (9%) had >1 mutation in > or =1 gene, including 52 cases that were compound heterozygous with mutations in >1 gene. Of the 562 distinct mutations, 394 (70%) were missense, 428 (76%) were seen once, and 336 (60%) are novel, including 92 of 199 in KCNQ1, 159 of 226 in KCNH2, and 70 of 110 in SCN5A. CONCLUSION: This cohort increases the publicly available compendium of putative LQTS-associated mutations by >50%, and approximately one-third of the most recently detected mutations continue to be novel. Although control population data suggest that the great majority of these mutations are pathogenic, expert interpretation of genetic test results will remain critical for effective clinical use of LQTS genetic test results.

Novel mutation in the SCN5A gene associated with arrhythmic storm development during acute myocardial infarction.

BACKGROUND: Ventricular tachycardia (VT) and ventricular fibrillation (VF) complicating Brugada syndrome, a genetic disorder linked to SCN5A mutations, and VF complicating acute myocardial infarction (AMI) both have been linked to phase 2 reentry. OBJECTIVE: Given the mechanistic similarities in arrhythmogenesis, the purpose of this study was to examine the contribution of SCN5A mutations to VT/VF complicating AMI. METHODS: Nineteen consecutive patients developing VF during AMI were enrolled in the study. Wild-type (WT) and mutant SCN5A genes were coexpressed with SCN1B in TSA201 cells and studied using whole-cell patch clamp techniques. RESULTS: Among the cohort of 19 patients, one missense mutation (G400A) in SCN5A was detected in a conserved region. An H558R polymorphism was detected on the same allele. Unlike the other 18 patients, who each developed 1-2 VF episodes during AMI, the mutation carrier developed six episodes of VT/VF within the first 12 hours. All VT/VF episodes were associated with ST-segment changes and were initiated by short-coupled extrasystoles. Flecainide and adenosine challenge performed to unmask Brugada and long QT syndromes both were negative. Peak G400A and G400A+H558R current were 70.7% and 88.4% less than WT current at -35 mV (P

Provocation of sudden heart rate oscillation with adenosine exposes abnormal QT responses in patients with long QT syndrome: a bedside test for diagnosing long QT syndrome.

AIMS: As arrhythmias in the long QT syndrome (LQTS) are triggered by heart rate deceleration or acceleration, we speculated that the sudden bradycardia and subsequent tachycardia that follow adenosine injection would unravel QT changes of diagnostic value in patients with LQTS. METHODS AND RESULTS: Patients (18 LQTS and 20 controls) received intravenous adenosine during sinus rhythm. Adenosine was injected at incremental doses until atrioventricular block or sinus pauses lasting 3 s occurred. The QT duration and morphology were studied at baseline and at the time of maximal bradycardia and subsequent tachycardia. Despite similar degree of adenosine-induced bradycardia (longest R-R 1.7+/-0.7 vs. 2.2+/-1.3 s for LQTS and controls, P=NS), the QT interval of LQT patients increased by 15.8+/-13.1%, whereas the QT of controls increased by only 1.5+/-6.7% (P<0.001). Similarly, despite similar reflex tachycardia (shortest R-R 0.58+/-0.07 vs. 0.55+/-0.07 s for LQT patients and controls, P=NS), LQTS patients developed greater QT prolongation (QTc=569+/-53 vs. 458+/-58 ms for LQT patients and controls, P<0.001). The best discriminator was the QTc during maximal bradycardia. Notched T-waves were observed in 72% of LQT patients but in only 5% of controls during adenosine-induced bradycardia (P<0.001). CONCLUSION: By provoking transient bradycardia followed by sinus tachycardia, this adenosine challenge test triggers QT changes that appear to be useful in distinguishing patients with LQTS from healthy controls.

Identification of genes regulated by chronic psychosocial stress and antidepressant treatment in the hippocampus.

Analysis of differentially expressed genes in the brain is a promising tool for elucidating pathological mechanisms that lead to central nervous disorders. Stress is known to be involved in the development of psychopathologies such as depression. In the present study, we searched for differentially expressed genes in the hippocampal formation after chronic psychosocial stress and after treatment with the antidepressant clomipramine. Experiments were conducted in male tree shrews, a valid psychosocial stress model in which antidepressant drugs prevent diverse effects of stress. Because many effects of stress have been attributed to the stress-induced elevation in glucocorticoids, we screened two subtractive hippocampal cDNA libraries generated from RNA of chronic cortisol-treated animals. Using real-time PCR to measure mRNA amounts, we identified five sequences whose expression levels differed between stressed animals and controls. Transcript levels of four of them, nerve growth factor (NGF), membrane glycoprotein 6a (M6a), CDC-like kinase 1 (CLK-1) and G-protein alpha q (GNAQ) were reduced by chronic psychosocial stress. Reduced amounts of these genes, which are all related to processes of cell differentiation, is in agreement with previous findings showing a retraction of dendrites and an impairment of neurogenesis in the hippocampal formation after chronic stress. An additional expressed sequence that was also regulated by stress could not be assigned to any known gene. Treatment with the antidepressant clomipramine prevented stress effects on expression of M6a, CLK-1, GNAQ and the novel sequence, but showed no effect on NGF stress-induced down-regulation. These findings support the concept that depressive disorders are accompanied by processes of neuronal dedifferentiation, at least in the hippocampal formation, and that antidepressants prevent these processes.

Sudden death associated with short-QT syndrome linked to mutations in HERG.

BACKGROUND: Sudden cardiac death takes the lives of more than 300 000 Americans annually. Malignant ventricular arrhythmias occurring in individuals with structurally normal hearts account for a subgroup of these sudden deaths. The present study describes the genetic basis for a new clinical entity characterized by sudden death and short-QT intervals in the ECG. METHODS AND RESULTS: Three families with hereditary short-QT syndrome and a high incidence of ventricular arrhythmias and sudden cardiac death were studied. In 2 of them, we identified 2 different missense mutations resulting in the same amino acid change (N588K) in the S5-P loop region of the cardiac IKr channel HERG (KCNH2). The mutations dramatically increase IKr, leading to heterogeneous abbreviation of action potential duration and refractoriness, and reduce the affinity of the channels to IKr blockers. CONCLUSIONS: We demonstrate a novel genetic and biophysical mechanism responsible for sudden death in infants, children, and young adults caused by mutations in KCNH2. The occurrence of sudden cardiac death in the first 12 months of life in 2 patients suggests the possibility of a link between KCNH2 gain of function mutations and sudden infant death syndrome. KCNH2 is the binding target for a wide spectrum of cardiac and noncardiac pharmacological compounds. Our findings may provide better understanding of drug interaction with KCNH2 and have implications for diagnosis and therapy of this and other arrhythmogenic diseases.

Differential regulation of polysialyltransferase expression during hippocampus development: Implications for neuronal survival.

Polysialyltransferases ST8SiaII/STX and ST8SiaIV/PST add polysialic acid (PSA) to the neural cell adhesion molecule (NCAM). Surface-located PSA is involved in cell-cell interactions participating in structural and functional plasticity of neuronal circuits. This study was undertaken to investigate the polysialyltransferase regulation pattern during hippocampal development. Polysialyltransferase expression levels analyzed by real-time RT-PCR indicated that ST8SiaII/STX mRNA is markedly down-regulated in vivo, decreasing abruptly at about the first week of postnatal development. ST8SiaII/STX mRNA is also down-regulated in hippocampal cells in culture, accompanying the morphological differentiation of neuronal interconnectivity. In contrast, ST8SiaIV/PST levels remain comparatively low during hippocampus ontogeny. Immunolabeling of primary hippocampal culture assays demonstrated that PSA expression parallels ST8SiaII/STX mRNA levels. In comparison, polysialyltransferase mRNA levels are not regulated in neuroblastoma cells during their proliferation. Sequence analysis of the 3'-untranslated region of ST8SiaII/STX cDNA indicated putative regulatory motifs. This information and the observed changes in mRNA half-life during development suggest that ST8SiaII/STX might be also regulated at the posttranscriptional level. To understand the reasons for the tight control of ST8SiaII/STX expression during development, we overexpressed the enzyme in hippocampal primary cultures by transfection. Overexpression of ST8SiaII/STX wild type as well as of a mutant lacking enzymatic activity affected neuronal viability, leading to cell death. However, this phenomenon was abolished by a double mutation in the ST8SiaII/STX that prevents formation of its three-dimentional structure. Interestingly, the overexpressed polysialyltransferase accumulates not only in the perinuclear region but also in the plasma membrane. Thus, overexpression of an ST8SiaII/STX that conserves its structure leads to abnormal accumulation of the protein, probably on the neuronal surface, affecting cell viability. This result explains the importance of an accurate regulation of polysialyltransferase expression during development.

Organoplatinum(II) complexes as a color biomarker in solid-phase peptide chemistry and screening.

[reaction: see text] A novel organoplatinum(II) biomarker is introduced to facilitate the solid-phase screening of combinatorial libraries for substrates and inhibitors of enzymes and receptors. The robust organoplatinum(II) biomarker can be incorporated, on amine functions, in peptides using standard peptide coupling techniques. The chemistry, stability, and (reversible) coloration process with KI(3) of the organoplatinum(II) biomarker was investigated.

Inhibition of nitric oxide synthesis aggravates reperfusion injury after hepatic ischemia and endotoxemia.

The potential role of nitric oxide (NO) was investigated in the pathophysiology of liver injury after priming with 20 min hepatic ischemia-reperfusion and administration of .5 mg/kg Salmonella enteritidis endotoxin. Liver injury during the early reperfusion phase of 4 h was characterized by severe vascular oxidant stress, lipid peroxidation (LPO), neutrophil infiltration, and a 33% reduction of the microvascular blood flow in the liver. Inhibition of NO synthesis with N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) aggravated liver injury by 90%, reduced LPO, and did not affect liver neutrophils but further impaired microvascular blood flow. Treatment with the NO-donor spermine-NONOate or L-arginine did not affect these parameters in postischemic animals, however, treatment did restore all values of L-NAME-treated animals back to disease control levels. These data suggest that endogenous NO formation is sufficient to limit ischemic liver injury during reperfusion but inhibition of NO synthesis will result in additional ischemic damage. NO may also be involved in scavenging of superoxide in the vasculature and in inducing LPO.

Heparan sulfate proteoglycan expression in normal human liver.

Because increasing evidence implicates heparan sulfate proteoglycans (HSPGs) as essential cofactors in receptor-growth factor interactions, in cell-cell recognition systems, and in cell-matrix adhesion processes and yet little is known about their cellular distribution pattern and cellular sources in liver tissue, we used monoclonal antibodies specific for the core proteins of syndecan1, 2, 3, 4, glypican, and perlecan to investigate their immunohistochemical expression in normal adult human liver biopsy specimens. Syndecan1 was expressed in sinusoidal endothelial cells, whereas the endothelium of the portal tract vessels was negative. Hepatocytes showed a membranous staining pattern of the sinusoidal and intercellular domain. Bile duct epithelial cells showed basolateral membrane positivity. Immunoreactivity for syndecan2 was seen in mesenchymal cells, accentuated around bile ducts. Syndecan3 showed intense staining of hepatic arterial and portal venous endothelial cells, of mesenchymal cells, and of Ito cells. Immunohistochemistry for syndecan4 showed a granular staining pattern of hepatocytes at their bile canalicular pole. Glypican showed weak positivity in portal tract mesenchymal cells and clear positivity in nerve bundles. Perlecan was present in Disse's space, in endothelial cells, in basement membranes surrounding bile ducts and vessels, in vessel walls, and in mesenchymal cells. The highly differential expression of these HSPGs in the different cell compartments of the liver, as well as in basement membranes and in Disse's space, suggests that each of these proteoglycans has a specific function in the interplay of cells, matrix molecules, growth factors, and proteinases.