Divergent Biochemical Properties and Disparate Impact of Arrhythmogenic Calmodulin Mutations on Zebrafish Cardiac Function.

Calmodulin (CaM) is a ubiquitous, small cytosolic calcium (Ca2+)-binding sensor that plays a vital role in many cellular processes by binding and regulating the activity of over 300 protein targets. In cardiac muscle, CaM modulates directly or indirectly the activity of several proteins that play a key role in excitation-contraction coupling (ECC), such as ryanodine receptor type 2 (RyR2),  l-type Ca2+ (Cav1.2), sodium (NaV1.5) and potassium (KV7.1) channels. Many recent clinical and genetic studies have reported a series of CaM mutations in patients with life-threatening arrhythmogenic syndromes, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT). We recently showed that four arrhythmogenic CaM mutations (N98I, D132E, D134H, and Q136P) significantly reduce the binding of CaM to RyR2. Herein, we investigate in vivo functional effects of these CaM mutations on the normal zebrafish embryonic heart function by microinjecting complementary RNA corresponding to CaMN98I, CaMD132E, CaMD134H, and CaMQ136P mutants. Expression of CaMD132E and CaMD134H mutants results in significant reduction of the zebrafish heart rate, mimicking a severe form of human bradycardia, whereas expression of CaMQ136P results in an increased heart rate mimicking human ventricular tachycardia. Moreover, analysis of cardiac ventricular rhythm revealed that the CaMD132E and CaMN98I zebrafish groups display an irregular pattern of heart beating and increased amplitude in comparison to the control groups. Furthermore, circular dichroism spectroscopy experiments using recombinant CaM proteins reveals a decreased structural stability of the four mutants compared to the wild-type CaM protein in the presence of Ca2+. Finally, Ca2+-binding studies indicates that all CaM mutations display reduced CaM Ca2+-binding affinities, with CaMD132E exhibiting the most prominent change. Our data suggest that CaM mutations can trigger different arrhythmogenic phenotypes through multiple and complex molecular mechanisms.

Development of Recombinant PLC-Zeta Protein as a Therapeutic Intervention for the Clinical Treatment of Oocyte Activation Failure.

The sperm-specific phospholipase C zeta (PLCζ) protein is widely considered as the predominant physiological stimulus for initiating the Ca2+ release responsible for oocyte activation during mammalian fertilization. The increasing number of genetic and clinical reports that directly link PLCζ defects and/or deficiencies with oocyte activation failure (OAF) necessitates the use of a powerful therapeutic intervention to overcome such cases of male factor infertility. Currently, in vitro fertilization (IVF) clinics treat OAF cases after intracytoplasmic sperm injection (ICSI) with Ca2+ ionophores. Despite their successful use, such chemical agents are unable to trigger the physiological pattern of Ca2+ oscillations. Moreover, the safety of these ionophores is not yet fully established. We have previously demonstrated that recombinant PLCζ protein can be successfully used to rescue failed oocyte activation, resulting in efficient blastocyst formation. Herein, we produced a maltose binding protein (MBP)-tagged recombinant human PLCζ protein capable of inducing Ca2+ oscillations in mouse oocytes similar to those observed at fertilization. Circular dichroism (CD) experiments revealed a stable, well-folded protein with a high helical content. Moreover, the recombinant protein could retain its enzymatic properties for at least up to 90 days after storage at -80 °C. Finally, a chick embryo model was employed and revealed that exposure of fertilized chicken eggs to MBP-PLCζ did not alter the embryonic viability when compared to the control, giving a first indication of its safety. Our data support the potential use of the MBP-PLCζ recombinant protein as an effective therapeutic tool but further studies are required prior to its use in a clinical setting.

Pathogenic Homocystinuria-Associated T236N Mutation Dramatically Alters the Biochemical Properties of Cystathionine Beta-Synthase Protein.

BACKGROUND: Cystathione beta-synthase (CBS) T236N is a novel mutation associated with pyridoxine non-responsiveness, which presents a significant difficulty in the medical treatment of homocystinuria. Reported severe phenotypes in homocystinuria patients highlight the urgent requirement to comprehend the molecular mechanisms underlying mutation pathogenicity for the advancement of the disease. METHODOLOGY: In this study, we used a multidisciplinary approach to investigate the molecular properties of bacterially expressed and purified recombinant CBST236N protein, which we directly compared to those of the wild-type (CBSWT) protein. RESULTS: Our data revealed a profound impact of the p.T236N mutation on CBS enzymatic activity, with a dramatic reduction of ~96% compared to the CBSWT protein. Circular dichroism (CD) experiments indicated that the p.T236N mutation did not significantly alter the secondary structure of the protein. However, CD spectra unveiled distinct differences in the thermal stability of CBSWT and CBST236N mutant protein species. In addition, chemical denaturation experiments further highlighted that the CBSWT protein exhibited greater thermodynamic stability than the CBST236N mutant, suggesting a destabilizing effect of this mutation. CONCLUSIONS: Our findings provide an explanation of the pathogenicity of the p.T236N mutation, shedding light on its role in severe homocystinuria phenotypes. This study contributes to a deeper understanding of CBS deficiency and may improve the development of targeted therapeutic strategies for affected individuals.

Biochemical and structural impact of two novel missense mutations in cystathionine ß-synthase gene associated with homocystinuria.

Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine ß-synthase (CBS). CBS is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme in the transsulfuration pathway, responsible for combining serine with homocysteine to produce cystathionine, whose activity is enhanced by the allosteric regulator S-adenosylmethionine (SAM). CBS also plays a role in generating hydrogen sulfide (H2S), a gaseous signaling molecule with diverse regulatory functions within the vascular, nervous, and immune systems. In this study, we present the clinical and biochemical characterization of two novel CBS missense mutations that do not respond to pyridoxine treatment, namely c.689T > A (L230Q) and 215A > T (K72I), identified in a Chinese patient. We observed that the disease-associated K72I genetic variant had no apparent effects on the spectroscopic and catalytic properties of the full-length enzyme. In contrast, the L230Q variant expressed in Escherichia coli did not fully retain heme and when compared with the wild-type enzyme, it exhibited more significant impairments in both the canonical cystathionine-synthesis and the alternative H2S-producing reactions. This reduced activity is consistent with both in vitro and in silico evidence, which indicates that the L230Q mutation significantly decreases the overall protein's stability, which in turn, may represent the underlying cause of its pathogenicity.

BRCA1 and Its Vulnerable C-Terminal BRCT Domain: Structure, Function, Genetic Mutations and Links to Diagnosis and Treatment of Breast and Ovarian Cancer.

The BRCA1 is a tumor suppressor gene that encodes for the BRCA1 protein, which plays a vital role in DNA repair, cell cycle regulation, and the maintenance of genomic stability. The BRCA1 protein interacts with a variety of other proteins that play essential roles in gene regulation and embryonic development. It is a large protein composed of multiple domains. The C-terminal region of the BRCA1 protein consists of two BRCT domains connected by a short linker. The BRCT domains are crucial in protein-protein interactions as well as in DNA damage response and cell cycle regulation through their phosphoprotein binding modules that recognize the phosphorylated protein sequence motif of other kinases. Mutations within the BRCT domain can disrupt the normal function of BRCA1 and lead to an increased risk of developing breast and ovarian cancer. Herein, we explore the structural characteristics of BRCA1, focusing on the BRCT domain, its interactions with key cellular components, and its involvement in various cellular processes. In addition, the impact of BRCT domain mutations on breast and ovarian cancer susceptibility, prognosis, and treatment options is discussed. By providing a comprehensive understanding of the BRCT domain of BRCA1, this review aims to shed light on the role of this important domain in the pathogenesis and potential therapeutic approaches for breast and ovarian cancer.

Arrhythmia-Associated Calmodulin E105A Mutation Alters the Binding Affinity of CaM to a Ryanodine Receptor 2 CaM-Binding Pocket.

Calmodulin (CaM) is a small, multifunctional calcium (Ca2+)-binding sensor that binds and regulates the open probability of cardiac ryanodine receptor 2 (RyR2) at both low and high cytosolic Ca2+ concentrations. Recent isothermal titration calorimetry (ITC) studies of a number of peptides that correspond to different regions of human RyR2 showed that two regions of human RyR2 (3584-3602aa and 4255-4271aa) bind with high affinity to CaM, suggesting that these two regions might contribute to a putative RyR2 intra-subunit CaM-binding pocket. Moreover, a previously characterized de novo long QT syndrome (LQTS)-associated missense CaM mutation (E105A) which was identified in a 6-year-old boy, who experienced an aborted first episode of cardiac arrest revealed that this mutation dysregulates normal cardiac function in zebrafish by a complex mechanism that involves alterations in both CaM-Ca2+ and CaM-RyR2 interactions. Herein, to gain further insight into how the CaM E105A mutation leads to severe cardiac arrhythmia, we generated large quantities of recombinant CaMWT and CaME105A proteins. We then performed ITC experiments to investigate and compare the interactions of CaMWT and CaME105A mutant protein with two synthetic peptides that correspond to the two aforementioned human RyR2 regions, which we have proposed to contribute to the RyR2 CaM-binding pocket. Our data reveal that the E105A mutation has a significant negative effect on the interaction of CaM with both RyR2 regions in the presence and absence of Ca2+, highlighting the potential contribution of these two human RyR2 regions to an RyR2 CaM-binding pocket, which may be essential for physiological CaM/RyR2 association and thus channel regulation.

Metal-binding cyclodextrins: Synthesis and complexation with Zn2+ and Ga3+ cations towards antimicrobial applications.

Highly resistant bacteria producing metallo-ß-lactamases (MBLs) to evade ß-lactam antibiotics, constitute a major cause of life-threatening infections world-wide. MBLs exert their hydrolytic action via Zn2+ cations in their active center. Presently, there are no approved drugs to target MBLs and combat the associated antimicrobial resistance (AMR). Towards this issue, we have prepared a family of cyclodextrins substituted with iminodiacetic acid (IDA) on their narrow side, while the wider side is either unmodified or per-2,3-O-methylated. The molecules form strong coordination complexes with Zn2+ or Ga3+ cations in aqueous solution. Free and metal-complexed compounds have been thoroughly characterized regarding structures, pH-dependent ionization states, distribution of species in solution, pKa values and metal-binding constants. At neutral pH the multi-anionic hosts bind up to four Zn2+ or Ga3+ cations. In vitro, 50 µΜ of the compounds achieve complete re-sensitization of MBL-producing Gram-negative clinical bacterial strains resistant to the carbapenems imipenem and meropenem. Moreover, the radioactive complex [67Ga]Ga-ß-IDACYD prepared, displays high radiochemical purity, sufficient stability both overtime and in the presence of human plasma apo-transferrin, thus providing an invaluable tool for future biodistribution and pharmacokinetic studies of ß-IDACYDin vivo, prerequisites for the development of therapeutic protocols.

ApoM binds endotoxin contributing to neutralization and clearance by High Density Lipoprotein.

Background: HDL possesses anti-inflammatory properties, however, the exact mechanism is not fully understood. Endotoxin is a potent inducers of TLR4 signaling, leading to inflammatory mediators' release. It has been estimated that TLR4 recognizes about 5% of circulating lipopolysaccharide whereas 95% is cleared by plasma lipoproteins, mainly HDL. ApoM is required for HDL biogenesis and 95% of plasma ApoM is found associated with HDL, both are significantly reduced during sepsis. Aim: The aim of this study is to investigate whether ApoM binds endotoxin and contributes to anti-inflammatory activity of HDL. Methods: Isothermal Titration Calorimetry (ITC) was used to determine the binding of ultrapure E. coli LPS to the recombinant ApoM protein. Purified human HDL and recombinant ApoM was used to investigate LPS neutralization using human and murine macrophages and computational simulation was performed. Result: ApoM shows high affinity for E. coli LPS, forming 1:1 complexes with Kd values below 1 µΜ, as revealed by ITC. The binding process is strongly exothermic and enthalpy-driven (ΔrH = -36.5 kJ/mol), implying the formation of an extensive network of interactions between ApoM and LPS in the bound state. Computational simulation also predicted high-affinity binding between ApoM and E. coli LPS and the best scoring models showed E. coli LPS docking near the calyx of ApoM without blocking the pocket. The biological significance of this interaction was further demonstrated in macrophages where purified HDL neutralized an E. coli LPS effect and significantly reduced TNFα release from human THP-1 cells. Conclusion: ApoM binds LPS to facilitate endotoxin neutralization and clearance by HDL.

Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket.

Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca2+) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaMN98I, CaMD132E, CaMD134H and CaMQ136P) and we used a CaM-RyR2 co-immunoprecipitation and a [3H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [3H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca2+, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association.

SPERM FACTORS AND EGG ACTIVATION: The structure and function relationship of sperm PLCZ1.

In 2002, sperm-specific phospholipase C zeta1 (PLCZ1) was discovered and through these 20 years, it has been established as the predominant sperm oocyte-activating factor. PLCZ1 cRNA expression or direct protein microinjection into mammalian oocytes triggers calcium (Ca2+) oscillations indistinguishable from those observed at fertilization. The imperative role of PLCZ1 in oocyte activation is revealed by the vast number of human mutations throughout the PLCZ1 gene that have been identified and directly linked with certain forms of male infertility due to oocyte activation deficiency. PLCZ1 is the smallest PLC in size, comprising four N-terminal EF-hand domains, followed by X and Y catalytic domains, which are separated by the XY-linker, and ending with a C-terminal C2 domain. The EF hands are responsible for the high Ca2+ sensitivity of PLCZ1. The X and Y catalytic domains are responsible for the catalysis of the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] substrate to produce the Ca2+-mobilising messenger, inositol 1,4,5-trisphosphate (IP3), while the XY-linker plays multiple roles in the unique mode of PLCZ1 action. Finally, the C2 domain has been proposed to facilitate the anchoring of PLCZ1 to intracellular vesicles through its direct interactions with specific phosphoinositides. This review discusses recent advances in the structure and function relationship of PLCZ1 and the potential binding partners of this important sperm-specific protein in the sperm and oocyte. The unravelling of all the remaining hidden secrets of sperm PLCZ1 should help us to understand the precise mechanism of fertilization, as well as enabling the diagnosis and treatment of currently unknown forms of PLCZ1 -linked human infertility.

Essential Role of Sperm-Specific PLC-Zeta in Egg Activation and Male Factor Infertility: An Update.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus responsible for generating calcium (Ca2+) oscillations that induce egg activation and early embryonic development during mammalian fertilization. In the mammalian testis, PLCζ expression is detected at spermiogenesis following elongated spermatid differentiation. Sperm-delivered PLCζ induces Ca2+ release via the inositol 1,4,5-trisphosphate (InsP3) signaling pathway. PLCζ is the smallest known mammalian PLC isoform identified to date, with the simplest domain organization. However, the distinctive biochemical properties of PLCζ compared with other PLC isoforms contribute to its unique potency in stimulating cytosolic Ca2+ oscillations within mammalian eggs. Moreover, studies describing PLCζ "knockout" mouse phenotypes confirm the supreme importance of PLCζ at egg activation and monospermic fertilization in mice. Importantly, a number of clinical reports have highlighted the crucial importance of PLCζ in human fertilization by associating PLCζ deficiencies with certain forms of male factor infertility. Herein, we give an update on recent advances that have refined our understanding of how sperm PLCζ triggers Ca2 + oscillations and egg activation in mammals, while also discussing the nature of a potential "alternative" sperm factor. We summarise PLCζ localization in mammalian sperm, and the direct links observed between defective PLCζ protein in sperm and documented cases of male infertility. Finally, we postulate how this sperm protein can be used as a potential diagnostic marker, and also as a powerful therapeutic agent for treatment of certain types of male infertility due to egg activation failure or even in more general cases of male subfertility.

Arrhythmogenic calmodulin E105A mutation alters cardiac RyR2 regulation leading to cardiac dysfunction in zebrafish.

Calmodulin (CaM) is a universal calcium (Ca2+ )-binding messenger that regulates many vital cellular events. In cardiac muscle, CaM associates with ryanodine receptor 2 (RyR2) and regulates excitation-contraction coupling. Mutations in human genes CALM1, CALM2, and CALM3 have been associated with life-threatening heart disorders, such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia. A novel de novo LQTS-associated missense CaM mutation (E105A) was recently identified in a 6-year-old boy, who experienced an aborted first episode of cardiac arrest. Herein, we report the first molecular characterization of the CaM E105A mutation. Expression of the CaM E105A mutant in zebrafish embryos resulted in cardiac arrhythmia and increased heart rate, suggestive of ventricular tachycardia. In vitro biophysical and biochemical analysis revealed that E105A confers a deleterious effect on protein stability and a reduced Ca2+ -binding affinity due to loss of cooperativity. Finally, the CaM E105A mutation resulted in reduced CaM-RyR2 interaction and defective modulation of ryanodine binding. Our findings suggest that the CaM E105A mutation dysregulates normal cardiac function by a complex mechanism involving alterations in both CaM-Ca2+ and CaM-RyR2 interactions.

Orientation of Biotin-Binding Sites in Streptavidin Adsorbed onto the Surface of Polythiophene Films.

The orientation of biotin-binding sites of streptavidin adsorbed to thin films of three polythiophenes (PTs), namely, regioregular poly(3-hexylthiophene) (RP3HT), regiorandom poly(3-butylthiophene) (P3BT), and poly(3,3‴-didodecylquaterthiophene) (PQT12), has been investigated. Polymer films were examined prior to and after protein adsorption with atomic force microscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Principal component analysis (PCA) applied to ToF-SIMS data revealed subtle changes in surface chemistry of polymer films and orientation of adsorbed streptavidin. PCA resolved the surface alignment of alkyl side chains and differentiated the ToF-SIMS data for PQT12, RP3HT, and P3BT, verifying an amorphous morphology for P3BT and a semicrystalline one for PQT12 and RP3HT. After the characterization of the polymeric films, streptavidin adsorption from solutions with different protein concentrations (up to 300 µg/mL) has been conducted. The PCA results distinguished between amino acids characteristic for external regions of streptavidin molecules adsorbed to different PTs suggest that streptavidin adsorbed to PQT12 exposes molecular regions rich in tryptophan and tyrosine, which are components of the biotin-binding sites. The latter results were confirmed using biotin-labeled horse radish peroxidase to estimate the exposed binding sites of streptavidin adsorbed onto the different PT films. The analysis of streptavidin structure suggests that interaction between polythiophene film and dipole moment of streptavidin subunit is responsible for orientation of biotin-binding sites.

Hypertrophic cardiomyopathy-linked variants of cardiac myosin-binding protein C3 display altered molecular properties and actin interaction.

The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3 + V4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0-C2 protein fragments revealed that c-MYBPC3 variants alter the C0-C2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0-C2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c-MYBPC3 mutations.

Designed positively charged cyclodextrin hosts with enhanced binding of penicillins as carriers for the delivery of antibiotics: The case of oxacillin.

In an effort to identify the optimal cyclodextrin (CD) host for delivery of penicillins to mammalian cells that will also offer protection against ß-lactamase-induced hydrolysis, nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) have been employed to study the inclusion complexes formed in aqueous solution between designed CD derivatives and two aminopenicillins, ampicillin and amoxicillin, and two antistaphylococcal penicillins, methicillin and oxacillin. Anionic and cationic thioether-substituted-ß- and -γCD derivatives were thus synthesized and compared with the neutral, parent CDs for complexation with the penicillins. The synthesized derivatives were shown to present ∼20% elongated cavity space in solution. Moreover, the cationic ones are >98% protonated at physiological pH. The most efficient host was the positively charged octakis[6-(2-aminoethylthio)-6-deoxy]-γ-CD (γCys) that formed the strongest complex with oxacillin (Kb ∼1700M-1) in an enthalpically and entropically favorable process (ΔHb=-10.5kJ/mol,TΔSb=8.0kJ/mol). In vitro biological tests demonstrated that γCys reduces 2.3-fold the rate of hydrolysis of oxacillin in the presence of oxa-1 ß-lactamase while displaying cell crossing capability and efficient internalization into macrophages as well as a sufficiently safe cytotoxicity profile. Overall, γCys could be considered as a promising vehicle for protection and delivery of oxacillin.

Mutations in PLCδ1 associated with hereditary leukonychia display divergent PIP2 hydrolytic function.

Hereditary leukonychia is a rare genetic nail disorder characterized by distinctive whitening of the nail plate of all 20 nails. Hereditary leukonychia may exist as an isolated feature, or in simultaneous occurrence with other cutaneous or systemic pathologies. Associations between hereditary leukonychia and mutations in the gene encoding phospholipase C delta-1 (PLCδ1) have previously been identified. However, the molecular mechanisms underlying PLCδ1 mutations and hereditary leukonychia remain uncharacterized. In the present study, we introduced hereditary leukonychia-linked human PLCδ1 mutations (C209R, A574T and S740R) into equivalent residues of rat PLCδ1 (C188R, A553T and S719R), and investigated their effect on the biophysical and biochemical properties of the PLCδ1 protein. Our data suggest that these PLCδ1 mutations associated with hereditary leukonychia do not uniformly alter the enzymatic ability of this protein leading to loss/gain of function, but result in significantly divergent enzymatic properties. We demonstrate here for the first time the importance of PLC-mediated calcium (Ca2+ ) signalling within the manifestation of hereditary leukonychia. PLCδ1 is almost ubiquitous in mammalian cells, which may explain why hereditary leukonychia manifests in association with other systemic pathologies relating to keratin expression.

Comparative Experimental and Computational Study of Monoalkyl Chain Phosphatidylcholine-Containing Thermoresponsive Liposomes.

Liposomes containing lysophospholipids are intensively studied as drug delivery systems that are stable at normal body temperature but exhibit fast release of their drug load at slightly elevated temperatures. In this study, the stability and release properties of dipalmitoylglycerophosphocholine (DPPC)-based liposomes incorporating the commonly used lysophosphatidylocholine (lyso-PC), and a series of monoalkyl chain ether-linked phosphatidylcholine, i.e., the biologically relevant monoalkyl chain platelet activating factor (PAF) and its derivatives lyso-PAF and methyl-PAF, were investigated. To this end a series of PEGylated small unilamellar liposomes with DPPC:monoalkyl lipid compositions of 5% and 10% molar ratio were prepared and compared with regard to stability (37 °C) and release properties at elevated temperatures (38-43 °C). All systems were characterized with respect to size distribution, ζ-potential, and phase transition characteristics. The presence of ether-lipids endows liposomes with superior (∼10% increase) release properties at 5% incorporation compared to lyso-PC, while at 10% molar ratio the formulations do not differ significantly, the release being close to 90%. The findings are supported by atomistic molecular dynamics simulations that suggest a correlation between the enhanced permeability and increased penetration of water molecules within the bilayers with density fluctuations resulting from the increased area-per-lipid and the disorder of the lysolipids alkyl chains.

Biodosimetry for High-Dose Exposures Based on Dicentric Analysis in Lymphocytes Released from the G2-Block by Caffeine.

High-dose assessments using the conventional dicentric assay are essentially restricted to doses up to 5 Gy and only to lymphocytes that succeed to proceed to first post-exposure mitosis. Since G2-checkpoint activation facilitates DNA damage recognition and arrest of damaged cells, caffeine is used to release G2-blocked lymphocytes overcoming the mitotic index and dicentric yield saturation problems, enabling thus dicentric analysis even at high-dose exposures. Using the fluorescence in situ hybridization technique with telomere and centromere peptide nucleic acid probes, the released lymphocytes, identified as metaphases with decondensed chromosomes following 1.5 h caffeine treatment, show increased yield of dicentrics compared to that obtained in lymphocytes that reach metaphase without G2-checkpoint abrogation by caffeine. Here, a 3-h caffeine/colcemid co-treatment before harvesting at 55 h post-exposure is used so that the dicentric analysis using Giemsa staining is based predominantly on lymphocytes released from the G2-block, increasing thus dicentric yield and enabling construction of a dose-response calibration curve with improved precision of high-dose estimates.

Distinctive malfunctions of calmodulin mutations associated with heart RyR2-mediated arrhythmic disease.

Calmodulin (CaM) is a cytoplasmic calcium sensor that interacts with the cardiac ryanodine receptor (RyR2), a large Ca(2+) channel complex that mediates Ca(2+) efflux from the sarcoplasmic reticulum (SR) to activate cardiac muscle contraction. Direct CaM association with RyR2 is an important physiological regulator of cardiac muscle excitation-contraction coupling and defective CaM-RyR2 protein interaction has been reported in cases of heart failure. Recent genetic studies have identified CaM missense mutations in patients with a history of severe cardiac arrhythmogenic disorders that present divergent clinical features, including catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT syndrome (LQTS) and idiopathic ventricular fibrillation (IVF). Herein, we describe how two CPVT- (N54I & N98S) and three LQTS-associated (D96V, D130G & F142L) CaM mutations result in alteration of their biochemical and biophysical properties. Ca(2+)-binding studies indicate that the CPVT-associated CaM mutations, N54I & N98S, exhibit the same or a 3-fold reduced Ca(2+)-binding affinity, respectively, versus wild-type CaM, whereas the LQTS-associated CaM mutants, D96V, D130G & F142L, display more profoundly reduced Ca(2+)-binding affinity. In contrast, all five CaM mutations confer a disparate RyR2 interaction and modulation of [(3)H]ryanodine binding to RyR2, regardless of CPVT or LQTS association. Our findings suggest that the clinical presentation of CPVT or LQTS associated with these five CaM mutations may involve both altered intrinsic Ca(2+)-binding as well as defective interaction with RyR2.