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β- adrenergic stimulation causes cell signaling that targets modulation of calcium levels as well as sarcomeric proteins to increases contractility. PKA phosphorylation of serine residues 23 and 24 of troponin I reduces calcium sensitivity and promotes cardiac relaxation. Our protein-protein docking and molecular dynamics studies revealed that Tpm-R160 is adjacent to these phosphorylation sites. In addition, Tpm-R160 has been identified as a disease-causing mutation site. Here, we investigated Tpm-R160's role in thin filament regulation and its interaction with phosphorylated TnI. Using in vitro motility assays, calcium sensitivity was quantified in reconstituted thin filaments containing various combinations of a phosphomimetic version of troponin I (TnI-S23/24D) and tropomyosin where the charge and potential for electrostatic interaction was removed by mutation of Tpm-R160 to a neutral alanine (Tpm-R160A). As expected, the phosphomimetic TnI (TnI-S23/24D) reduced calcium sensitivity in thin filaments with wild-type tropomyosin. However, the phosphorylation-like effect was diminished in the presence of the Tpm-R160A mutation. Notably, Tpm-R160A alone, when paired with wild-type TnI, also showed reduced calcium sensitivity, indicating that mutation of Tpm-R160 affects thin filament regulation in the absence of phosphorylation. Our findings indicate that Tpm-R160 has a dual role in thin filament regulation: (1)it is crucial for proper interaction between phosphorylated TnI and Tpm-R160 during adrenergic-induced cardiac relaxation , and (2) at the same time, the arginine residue itself is additionally required for maintenance of overall calcium sensitivity. These results provide key insight into the molecular defects underlying cardiomyopathy and a framework for development of therapeutic strategies targeting Tpm-R160 interactions. (249 words)., Competing Interests: Declaration of Competing Interest The authors do not have any financial or other substantive conflicts of interest that could be construed to influence the results or interpretation of the data presented., (Copyright © 2025. Published by Elsevier Inc.)

Sarcomere activation in striated muscle requires both thin filament-based and thick filament-based activation mechanisms. Recent studies have shown that myosin heads on the thick filaments undergo OFF to ON structural transitions in response to calcium (Ca2+) in permeabilized porcine myocardium in the presence of a small molecule inhibitor that eliminated active force. The changes in X-ray diffraction signatures of OFF to ON transitions were interpreted as Ca2+ acting to activate the thick filaments. Alternatively, Ca2+ binding to troponin could initiate a Ca2+-dependent crosstalk from the thin filament to the thick filament via interfilament connections such as the myosin binding protein-C. Here, we exchanged native troponin in permeabilized porcine myocardium for troponin containing the cTnC D65A mutation, which disallows the activation of troponin through Ca2+ binding to determine if Ca2+-dependent thick filament activation persists in the absence of thin filament activation. After the exchange protocol, over 95% of the Ca2+-activated force was eliminated. Equatorial intensity ratio increased significantly in both WT and D65A exchanged myocardium with increasing Ca2+ concentration. The degree of helical ordering of the myosin heads decreased by the same amount in WT and D65A myocardium when Ca2+ concentration increased. These results are consistent with a direct effect of Ca2+ in activating the thick filament rather than an indirect effect due to Ca2+-mediated crosstalk between the thick and thin filaments., (© 2024 Mohran et al.)

Dog and cat preference has been associated with a few factors, like owner personality traits, but data regarding other aspects of preference ontogeny and the impact of preferences on pet wellbeing have yet to be examined. In this exploratory study, several of these characteristics, such as exposure to pets when young and as adults and current pet interactions and diet were analyzed from internet survey data. We found that more people identified as dog people (63.3%) versus cat people (36.7%) and preference for dogs remained consistent from childhood to adulthood compared with cats. In individuals who changed species preference, a lack of childhood exposure to cats (47.2%) was significantly associated with the group that changed preferences from dogs to cats from childhood to adulthood, compared with dog ownership as a child in the group that changed preferences from cats to dogs (24.4%). The number of cats and dogs in the home directly correlated with species preference ( p < 0.001). Dwelling location was also significantly associated with species preference, with cat people being more likely to live in an urban area and dog people in a rural area ( p = 0.002). More time was spent in both active and passive interactions with pets of the preferred species. Cats owned by cat people were more likely to be fed prescription diets compared with cats owned by dog people ( p < 0.001). Interestingly, dog people were more likely to feed both their cats ( p = 0.012) and dogs ( p < 0.001) a raw diet compared with cat people. Additional research is needed to understand the development and impact of owner species preferences on pets to identify risks of suboptimal wellbeing.

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis, we set out to determine whether SoxB1 transcription factors have a regulatory function in NPB formation. Here, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq, we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as potential Sox3 partners in regulating the formation of the NPB and show that their combined activity is needed for normal NPB gene expression. Together, these data identify a role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (K i , 0.32 and 1.76 μM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

SoxB1 transcription factors (Sox2/3) are well known for their role in early neural fate specification in the embryo, but little is known about functional roles for SoxB1 factors in non-neural ectodermal cell types, such as the neural plate border (NPB). Using Xenopus laevis , we set out to determine if SoxB1 transcription factors have a regulatory function in NPB formation. Herein, we show that SoxB1 factors are necessary for NPB formation, and that prolonged SoxB1 factor activity blocks the transition from a NPB to a neural crest state. Using ChIP-seq we demonstrate that Sox3 is enriched upstream of NPB genes in early NPB cells and, surprisingly, in blastula stem cells. Depletion of SoxB1 factors in blastula stem cells results in downregulation of NPB genes. Finally, we identify Pou5f3 factors as a potential SoxB1 partners in regulating the formation of the NPB and show their combined activity is needed to maintain NPB gene expression. Together, these data identify a novel role for SoxB1 factors in the establishment and maintenance of the NPB, in part through partnership with Pou5f3 factors.

Background: Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking., Methods: Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficit., Results: Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the ON state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ., Conclusions: As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy., Competing Interests: Disclosures None.

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the "on" state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Danicamtiv corrected calcium sensitivity in demembranated and abnormal twitch magnitude and kinetics in intact cardiac tissue., Significance Statement: Directly augmenting sarcomere function has potential to overcome limitations of currently used inotropic agents to improve cardiac contractility. Myosin modulation is a novel mechanism for increased contraction in cardiomyopathies. Danicamtiv is a myosin activator that is currently under investigation for use in cardiomyopathy patients. Our study is the first detailed mechanism of how Danicamtiv increases force and alters kinetics of cardiac activation and relaxation. This new understanding of the mechanism of action of Danicamtiv can be used to help identify patients that could benefit most from this treatment.

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ∼1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to modulate myosin states to enhance contractility and induce metabolic flexibility to support improved function in CMs., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Study Objectives: This study examines the impact of cognitive behavioral therapy for insomnia (CBT-I) and positive airway pressure (PAP) therapy for comorbid insomnia and sleep apnea on nocturnal sleep and daytime functioning., Methods: A partial factorial design was used to examine treatment pathways with CBT-I and PAP and the relative benefits of each treatment. One hundred eighteen individuals with comorbid insomnia and sleep apnea were randomized to receive CBT-I followed by PAP, self-monitoring followed by CBT-I concurrent with PAP, or self-monitoring followed by PAP only. Participants were assessed at baseline, PAP titration, and 30 and 90 days after PAP initiation. Outcome measures included sleep diary- and actigraphy-measured sleep, Flinders Fatigue Scale, Epworth Sleepiness Scale, Functional Outcome of Sleep Questionnaire, and cognitive emotional measures., Results: A main effect of time was found on diary-measured sleep parameters (decreased sleep onset latency and wake after sleep onset; increased total sleep time and sleep efficiency) and actigraphy-measured sleep parameters (decreased wake after sleep onset; increased sleep efficiency) and daytime functioning (reduced Epworth Sleepiness Scale, Flinders Fatigue Scale; increased Functional Outcome of Sleep Questionnaire) across all arms (all P < .05). Significant interactions and planned contrast comparisons revealed that CBT-I was superior to PAP and self-monitoring on reducing diary-measured sleep onset latency and wake after sleep onset and increasing sleep efficiency, as well as improving Functional Outcome of Sleep Questionnaire and Flinders Fatigue Scale compared to self-monitoring., Conclusions: Improvements in sleep and daytime functioning were found with PAP alone or concomitant with CBT-I. However, more rapid effects were observed on self-reported sleep and daytime performance when receiving CBT-I regardless of when it was initiated. Therefore, concomitant treatment appears to be a favorable approach to accelerate treatment outcomes., Clinical Trial Registration: Registry: ClinicalTrials.gov; Name: Multidisciplinary Approach to the Treatment of Insomnia and Comorbid Sleep Apnea (MATRICS); URL: https://clinicaltrials.gov/ct2/show/NCT01785303; Identifier: NCT01785303., Citation: Tu AY, Crawford MR, Dawson SC, et al. A randomized controlled trial of cognitive behavioral therapy for insomnia and PAP for obstructive sleep apnea and comorbid insomnia: effects on nocturnal sleep and daytime performance. J Clin Sleep Med . 2022;18(3):789-800., (© 2022 American Academy of Sleep Medicine.)

Purpose/background: Attentional bias toward drug-related cues is considered to be an indication of neurocognitive processes associated with drug dependence. While this phenomenon has been shown in other addictive substances, whether hypnotic medication would lead to similar processes remains an issue to be investigated. The present study examined attentional bias toward drug-related cues in long-term hypnotic users and the effect of negative affect on this process., Methods/procedures: Thirteen long-term hypnotic users participated in this study. They spent 2 nights in the sleep laboratory: a mood-induction night and a neutral night. Attentional bias was measured through the recording of event-related potentials using a cue-reactivity paradigm; subjective craving for hypnotics was assessed using a single-item rating scale, and negative affect was measured using the Positive and Negative Affect Schedule., Findings/results: The results showed that the amplitudes of P300 and slow positive wave for hypnotic-related and sleep-related photographs were significantly higher than those for neutral photographs in both conditions. Negative mood induction did not significantly increase attentional bias., Implications/conclusions: The findings provide preliminary evidence that long-term hypnotic users do have attentional bias for hypnotic-related photos, suggesting the possibility of neurocognitive processes associated with drug dependence. However, the results did not show higher attentional bias under negative mood, suggesting that the use of hypnotics is not reinforced by the desire to eliminate negative affect. Because of the limited sample size and lack of a control group, the results should be considered as preliminary findings that call for future studies to further investigate this issue.

The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet-binding A1 domain of VWF is connected to the VWF N-terminally located D'D3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ibα (GpIbα). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated. Here, we show that an aspartate at position 1261 is the most critical residue of the N-terminal linker for inhibiting binding of the VWF A1 domain to GpIbα on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1-GpIbα binding experiments, we identified a network of salt bridges between Asp 1261 and the rest of A1 that lock the N-terminal linker in place such that it reduces binding to GpIbα. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIbα, in which case the mutations inhibited the binding. These results show that interactions between charged amino acid residues are important both to directly stabilize the A1-GpIbα complex and to indirectly destabilize the complex through the N-terminal linker., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

cTnI(P82S) (cTnI(P83S) in rodents) resides at the I-T arm of cardiac troponin I (cTnI) and was initially identified as a disease-causing mutation of hypertrophic cardiomyopathy (HCM). However, later studies suggested this may not be true. We recently reported that introduction of an HCM-associated mutation in either inhibitory-peptide (cTnI(R146G)) or cardiac-specific N-terminus (cTnI(R21C)) of cTnI blunts the PKA-mediated modulation on myofibril activation/relaxation kinetics by prohibiting formation of intrasubunit contacts between these regions. Here, we tested whether this also occurs for cTnI(P83S). cTnI(P83S) increased both Ca(2+) binding affinity to cTn (KCa) and affinity of cTnC for cTnI (KC-I), and eliminated the reduction of KCa and KC-I observed for phosphorylated-cTnI(WT). In isolated myofibrils, cTnI(P83S) maintained maximal tension (TMAX) and Ca(2+) sensitivity of tension (pCa50). For cTnI(WT) myofibrils, PKA-mediated phosphorylation decreased pCa50 and sped up the slow-phase relaxation (especially for those Ca(2+) conditions that heart performs in vivo). Those effects were blunted for cTnI(P83S) myofibrils. Molecular-dynamics simulations suggested cTnI(P83S) moderately inhibited an intrasubunit interaction formation between inhibitory-peptide and N-terminus, but this "blunting" effect was weaker than that with cTnI(R146G) or cTnI(R21C). In summary, cTnI(P83S) has similar effects as other HCM-associated cTnI mutations on troponin and myofibril function even though it is in the I-T arm of cTnI.

Two hypertrophic cardiomyopathy-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory peptide and the cardiac-specific N terminus. We recently reported that these regions may interact when Ser-23/Ser-24 are phosphorylated, weakening the interaction of cTnI with cardiac TnC. Little is known about how these mutations influence the affinity of cardiac TnC for cTnI (KC-I) or contractile kinetics during β-adrenergic stimulation. Here, we tested how cTnI(R146G) or cTnI(R21C) influences contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca(2+) binding affinity to cTn (KCa) and KC-I. PKA phosphorylation resulted in a similar reduction of KCa for all complexes, but KC-I was reduced only with cTnI(WT). cTnI(WT), cTnI(R146G), and cTnI(R21C) were complexed into cardiac troponin and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (Tmax) was maintained for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils, and Ca(2+) sensitivity of tension (pCa50) was increased. PKA phosphorylation decreased pCa50 for cTnI(WT)-exchanged myofibrils but not for either mutation. PKA phosphorylation accelerated the early slow phase relaxation for cTnI(WT) myofibrils, especially at Ca(2+) levels that the heart operates in vivo. Importantly, this effect was blunted for cTnI(R146G)- and cTnI(R21C)-exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N terminus and the inhibitory peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together, our results suggest that cTnI(R146G) and cTnI(R21C) blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory peptide., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The ends of coiled-coil tropomyosin molecules are joined together by nine to ten residue-long head-to-tail "overlapping domains". These short four-chained interconnections ensure formation of continuous tropomyosin cables that wrap around actin filaments. Molecular Dynamics simulations indicate that the curvature and bending flexibility at the overlap is 10-20% greater than over the rest of the molecule, which might affect head-to-tail filament assembly on F-actin. Since the penultimate residue of striated muscle tropomyosin, Ser283, is a natural target of phosphorylating enzymes, we have assessed here if phosphorylation adjusts the mechanical properties of the tropomyosin overlap domain. MD simulations show that phosphorylation straightens the overlap to match the curvature of the remainder of tropomyosin while stiffening it to equal or exceed the rigidity of canonical coiled-coil regions. Corresponding EM data on phosphomimetic tropomyosin S283D corroborate these findings. The phosphorylation-induced change in mechanical properties of tropomyosin likely results from electrostatic interactions between C-terminal phosphoSer283 and N-terminal Lys12 in the four-chain overlap bundle, while promoting stronger interactions among surrounding residues and thus facilitating tropomyosin cable assembly. The stiffening effect of D283-tropomyosin noted correlates with previously observed enhanced actin-tropomyosin activation of myosin S1-ATPase, suggesting a role for the tropomyosin phosphorylation in potentiating muscle contraction., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Many current pharmaceutical therapies for systolic heart failure target intracellular [Ca(2+)] ([Ca(2+)]i) metabolism, or cardiac troponin C (cTnC) on thin filaments, and can have significant side-effects, including arrhythmias or adverse effects on diastolic function. In this study, we tested the feasibility of directly increasing the Ca(2+) binding properties of cTnC to enhance contraction independent of [Ca(2+)]i in intact cardiomyocytes from healthy and myocardial infarcted (MI) hearts. Specifically, cardiac thin filament activation was enhanced through adenovirus-mediated over-expression of a cardiac troponin C (cTnC) variant designed to have increased Ca(2+) binding affinity conferred by single amino acid substitution (L48Q). In skinned cardiac trabeculae and myofibrils we and others have shown that substitution of L48Q cTnC for native cTnC increases Ca(2+) sensitivity of force and the maximal rate of force development. Here we introduced L48Q cTnC into myofilaments of intact cardiomyocytes via adeno-viral transduction to deliver cDNA for the mutant or wild type (WT) cTnC protein. Using video-microscopy to monitor cell contraction, relaxation, and intracellular Ca(2+) transients (Fura-2), we report that incorporation of L48Q cTnC significantly increased contractility of cardiomyocytes from healthy and MI hearts without adversely affecting Ca(2+) transient properties or relaxation. The improvements in contractility from L48Q cTnC expression are likely the result of enhanced contractile efficiency, as intracellular Ca(2+) transient amplitudes were not affected. Expression and incorporation of L48Q cTnC into myofilaments was confirmed by Western blot analysis of myofibrils from transduced cardiomyocytes, which indicated replacement of 18±2% of native cTnC with L48Q cTnC. These experiments demonstrate the feasibility of directly targeting cardiac thin filament proteins to enhance cardiomyocyte contractility that is impaired following MI., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Two cTnC variants, L57Q and I61Q, both of which are located on helix C within the N domain of cTnC, were originally reported in the skeletal muscle system [Tikunova, Davis, J. Biol. Chem. 279 (2004) 35341-35352], as the analogous L58Q and I62Q sTnC, and demonstrated a decreased Ca(2+) binding affinity. Here, we provide detailed characterization of structure-function relationships for these two cTnC variants, to determine if they behave differently in the cardiac system and as a framework for determining similarities and differences with other cTnC mutations that have been associated with DCM. We have used an integrative approach to study the structure and function of these cTnC variants both in solution and in silico, to understand how the L57Q and I61Q mutations influence Ca(2+) binding at site II, the subsequent effects on the interaction with cTnI, and the structural changes which are associated with these changes. Steady-state and stopped flow fluorescence spectroscopy confirmed that a decrease in Ca(2+) affinity for recombinant cTnC and cTn complexes containing the L57Q or I61Q variants. The L57Q variant was intermediate between WT and I61Q cTnC and also did not significantly alter cTnC-cTnI interaction in the absence of Ca(2+), but did decrease the interaction in the presence of Ca(2+). In contrast, I61Q decreased the cTnC-cTnI interaction in both the absence and presence of Ca(2+). This difference in the absence of Ca(2+) suggests a greater structural change in cNTnC may occur with the I61Q mutation than the L57Q mutation. MD simulations revealed that the decreased Ca(2+) binding induced by I61Q may result from destabilization of the Ca(2+) binding site through interruption of intra-molecular interactions when residue 61 forms new hydrogen bonds with G70 on the Ca(2+) binding loop. The experimentally observed interruption of the cTnC-cTnI interaction caused by L57Q or I61Q is due to the disruption of key hydrophobic interactions between helices B and C in cNTnC. This study provides a molecular basis of how single mutations in the C helix of cTnC can reduce Ca(2+) binding affinity and cTnC-cTnI interaction, which may provide useful insights for a better understanding of cardiomyopathies and future gene-based therapies., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Calcium sensitivity of the force-pCa relationship depends strongly on sarcomere length (SL) in cardiac muscle and is considered to be the cellular basis of the Frank-Starling law of the heart. SL dependence may involve changes in myofilament lattice spacing and/or myosin crossbridge orientation to increase probability of binding to actin at longer SLs. We used the L48Q cardiac troponin C (cTnC) variant, which has enhanced Ca(2+) binding affinity, to test the hypotheses that the intrinsic properties of cTnC are important in determining 1) thin filament binding site availability and responsiveness to crossbridge activation and 2) SL dependence of force in cardiac muscle. Trabeculae containing L48Q cTnC-cTn lost SL dependence of the Ca(2+) sensitivity of force. This occurred despite maintaining the typical SL-dependent changes in maximal force (F(max)). Osmotic compression of preparations at SL 2.0 μm with 3% dextran increased F(max) but not pCa(50) in L48Q cTnC-cTn exchanged trabeculae, whereas wild-type (WT)-cTnC-cTn exchanged trabeculae exhibited increases in both F(max) and pCa(50). Furthermore, crossbridge inhibition with 2,3-butanedione monoxime at SL 2.3 μm decreased F(max) and pCa(50) in WT cTnC-cTn trabeculae to levels measured at SL 2.0 μm, whereas only F(max) was decreased with L48Q cTnC-cTn. Overall, these results suggest that L48Q cTnC confers reduced crossbridge dependence of thin filament activation in cardiac muscle and that changes in the Ca(2+) sensitivity of force in response to changes in SL are at least partially dependent on properties of thin filament troponin.

Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein-protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca(2+) sensitivity of the thin filament have been linked to disease. Tikunova and Davis engineered a series of cNTnC mutations that altered Ca(2+) binding properties and studied the effects on the Ca(2+) sensitivity of the thin filament and contraction [Tikunova, S. B., and Davis, J. P. (2004) J. Biol. Chem. 279, 35341-35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in the cNTnC Ca(2+) binding affinity and Ca(2+) sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca(2+) sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca(2+) and cTnI to cTnC. Nuclear magnetic resonance chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC's conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca(2+) sensitivity can do so by modulating Ca(2+) and cTnI binding.

The hemostatic function of von Willebrand factor is downregulated by the metalloprotease ADAMTS13, which cleaves at a unique site normally buried in the A2 domain. Exposure of the proteolytic site is induced in the wild-type by shear stress as von Willebrand factor circulates in blood. Mutations in the A2 domain, which increase its susceptibility to cleavage, cause type 2A von Willebrand disease. In this study, molecular dynamics simulations suggest that the A2 domain unfolds under tensile force progressively through a series of steps. The simulation results also indicated that three type 2A mutations in the C-terminal half of the A2 domain, L1657I, I1628T and E1638K, destabilize the native state fold of the protein. Furthermore, all three type 2A mutations lowered in silico the tensile force necessary to undock the C-terminal helix α6 from the rest of the A2 domain, the first event in the unfolding pathway. The mutations F1520A, I1651A and A1661G were also predicted by simulations to destabilize the A2 domain and facilitate exposure of the cleavage site. Recombinant A2 domain proteins were expressed and cleavage assays were performed with the wild-type and single-point mutants. All three type 2A and two of the three predicted mutations exhibited increased rate of cleavage by ADAMTS13. These results confirm that destabilization of the helix α6 in the A2 domain facilitates exposure of the cleavage site and increases the rate of cleavage by ADAMTS13.

Myosin binding protein C (MyBP-C) is a thick-filament protein that limits cross-bridge cycling rates and reduces myocyte power output. To investigate mechanisms by which MyBP-C affects contraction, we assessed effects of recombinant N-terminal domains of cardiac MyBP-C (cMyBP-C) on contractile properties of permeabilized rat cardiac trabeculae. Here, we show that N-terminal fragments of cMyBP-C that contained the first three immunoglobulin domains of cMyBP-C (i.e., C0, C1, and C2) plus the unique linker sequence termed the MyBP-C "motif" or "m-domain" increased Ca(2+) sensitivity of tension and increased rates of tension redevelopment (i.e., k(tr)) at submaximal levels of Ca(2+). At concentrations > or =20 microM, recombinant proteins also activated force in the absence of Ca(2+) and inhibited maximum Ca(2+)-activated force. Recombinant proteins that lacked the combination of C1 and the motif did not affect contractile properties. These results suggest that the C1 domain plus the motif constitute a functional unit of MyBP-C that can activate the thin filament.

The unique myosin binding protein-c "motif" near the N-terminus of myosin binding protein-C (MyBP-C) binds myosin S2. Previous studies demonstrated that recombinant proteins containing the motif and flanking regions (e.g., C1C2) affect thin filament movement in motility assays using heavy meromyosin (S1 plus S2) as the molecular motor. To determine if S2 is required for these effects we investigated whether C1C2 affects motility in assays using only myosin S1 as the motor protein. Results demonstrate that effects of C1C2 are comparable in both systems and suggest that the MyBP-C motif affects motility through direct interactions with actin and/or myosin S1.

Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of approximately 110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where filaments are maximally activated (i.e. either in the absence of thin filament regulatory proteins or in the presence of troponin and tropomyosin and high [Ca2+]). By contrast, under conditions where thin filament sliding speed is submaximal (i.e. in the presence of troponin and tropomyosin and low [Ca2+]), proteins containing the motif increased filament speed. Recombinant N-terminal proteins also bound to F-actin and inhibited acto-HMM ATPase rates in solution. The results suggest that N-terminal domains of MyBP-C slow cross-bridge cycling kinetics by reducing rates of cross-bridge detachment.

Human plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. In this study, we investigated the effects of lipoproteins on the secretion of PLTP in cultured BeWo choriocarcinoma cells. Low-density lipoproteins (LDLs) decreased PLTP secretion in a dose- and time-dependent manner, whereas very low density lipoproteins and high-density lipoproteins (HDLs) had little effect. LDL suppression of PLTP secretion was not altered by the inhibition of both LDL receptor and LDL receptor-related protein with receptor-associated protein. Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor, U0126, could abolish the LDL-mediated inhibition of PLTP secretion. Furthermore, LDL, but not HDL, could stimulate the expression of MAPK phosphatase-1 (MKP-1) in BeWo cells that resulted in the inactivation of p44/p42 extracellular signal-regulated kinase (ERK) 1 and 2, the family members of MAPKs. These results support the conclusion that LDL-mediated suppression of PLTP secretion in BeWo cells is through a LDL receptor-independent MAPK signaling pathway.

The number, amplitude, duration, and bout structure of isolation-induced ultrasonic vocalization (USV) of infant rats (Rattus norvegicus) were measured on postnatal Day 10. Measurements were made before and after a brief, 1-min, active interaction with their mother or before and after a "pick-up" control procedure. Consistent with prior studies, the number of USVs emitted was significantly increased in the period following the maternal reunion but not after the control procedure. The average amplitude of USVs was also greater following maternal reunion. Finally, analyses characterizing the bout structure of USV production indicated that the average bout size (i.e. number of USVs/bout) was increased severalfold following the reunion with the mother, accounting for the greater rate of USV production during the second isolation period., (((c) 2004 APA, all rights reserved))

Objective: Phospholipid transfer protein (PLTP), an important protein in the transfer of phospholipids between lipoprotein particles and in the remodeling of HDL, is regulated at both the transcriptional and the protein level. We performed quantitative trait locus (QTL) analysis to identify genomic loci regulating PLTP activity in mice., Methods and Results: Plasma PLTP activity was measured in 217 male F2 progeny from a SM/J x NZB/B1NJ intercross. Two QTL for plasma PLTP activity in mice fed chow (Pltpq1 and Pltpq2) were found on chromosomes 3 (34 cM, logarithm of odds [LOD] 3.5) and 10 (66 cM, LOD 4.1); two additional QTL in mice fed atherogenic diet (Pltpq3 and Pltpq4) were found on chromosomes 9 (56 cM, LOD 4.5) and 15 (34 cM, LOD 5.0); and one QTL (Pltiq1) for the inducibility of PLTP activity was found on chromosome 4 (70 cM, LOD 3.7). Several candidate genes for these 5 QTL were tested by sequence comparison and expression studies., Conclusions: We identified five significant loci involved in PLTP activity in the mouse and provided supporting evidence for the candidacy of Nr1h4 and Apof as the genes underlying Pltpq2.

Background: Phospholipid transfer protein (PLTP) plays an important role in HDL particle metabolism and may modulate hepatic secretion of apolipoprotein B-containing lipoproteins. However, whether PLTP might participate directly in human atherosclerotic lesion formation is unknown., Methods and Results: The cellular and extracellular distributions of PLTP were determined in normal and atherosclerotic human coronary lesions with a monoclonal antibody to human PLTP. Cell types (smooth muscle cells [SMCs] or macrophages), apolipoproteins (apoA-I, apoB, and apoE), and extracellular matrix proteoglycans (biglycan and versican) were identified on adjacent sections with monospecific antibodies. Minimal extracellular PLTP was detected in nonatherosclerotic coronary arteries, but extracellular and cellular PLTP immunostaining was widespread in atherosclerotic lesions. PLTP was detected in foam cell SMCs and in foam cell macrophages, which suggests that cellular cholesterol accumulation might increase PLTP expression in both cell types. This was confirmed by in vitro studies demonstrating that cholesterol loading of macrophages leads to 2- to 3-fold increases in PLTP steady-state mRNA levels, protein expression, and activity. PLTP also was detected in an extracellular distribution, colocalizing with apoA-I, apoB, apoE, and the vascular proteoglycan biglycan. In gel mobility shift assays, both active and inactive recombinant PLTP markedly increased HDL binding to biglycan, which suggests that PLTP may mediate lipoprotein binding to proteoglycans independent of its phospholipid transfer activity., Conclusions: PLTP is present in human atherosclerotic lesions, and its distribution suggests roles for PLTP in both cellular cholesterol metabolism and lipoprotein retention on extracellular matrix.

Two experiments tested the effects of lactation and weaning on heart rate (HR), corticosterone, and behavioral responses to stress in Wistar rats. In Experiment 1, HRs in lactating, weaning, and control animals were recorded for 10-min periods before, during, and after immobilization stress. Compared with control animals, lactating and weaning animals showed a diminished initial HR response. In addition, HRs of weaning animals failed to habituate and showed a delayed decline after stress termination. In Experiment 2, behaviors, HRs, and corticosterone levels in the elevated plus maze (EPM) were compared among lactating, weaning, and control animals. Compared with control animals, weaning animals exhibited more anxiety behaviors. Contrary to expectation, compared to the other two groups, lactating animals exhibited more closed-arm entries, although they may have been motivated by maternal behavior, rather than anxiety. Initial HR responses to the plus maze were attenuated in lactating animals. Corticosterone levels after the plus maze were lowest in the lactating dams and highest in the control animals. The results from these two experiments are consistent with effects of breast-feeding and weaning observed in humans. In general, lactation is associated with an attenuated initial HR response to stress, while weaning is associated with exacerbated response to stressors.

Phospholipid transfer protein (PLTP) plays an important role in the metabolism of plasma high density lipoprotein. The mouse gene encoding PLTP and its promoter region has been cloned in our laboratory. The present study was conducted to functionally analyze the transcriptional regulation of the mouse PLTP gene. The results indicated that DNA sequences between -245 and -69 were responsible for the full promoter activity and binding motifs for transcription factor Sp1 and AP-2 within this functional promoter region were synergistically essential for the basal transcription. The transcriptional activity of this gene was significantly increased by chenodeoxycholic acid and fenofibrate, suggesting that transcription factor farnesoid X-activated receptor (FXR) and peroxisome proliferator-activated receptor (PPAR) are likely involved in the transcriptional regulation. DNA sequence analysis suggests that DNA sequences from -407 to -395 and from -393 to -381 are homologous to the recognition motifs of FXR, and those from -859 to -847 and from -309 to -297 are similar to the potential binding motif for PPAR. These findings provide a molecular basis for further investigation of the physiological function and regulation of the PLTP gene in mice., (Copyright 2001 Academic Press.)

Phospholipid transfer protein (PLTP) plays an important role in human plasma HDL metabolism. Clinical data have recently indicated that plasma PLTP activity and mass were both higher in diabetic patients concomitant with hyperglycemia. The present study shows that high glucose increases both PLTP mRNA and functional activity in HepG2 cells, due to a significant increase in the promoter activity of human PLTP gene. The glucose-responsive elements are located between -759 and -230 of the PLTP 5'-flanking region, within which two binding motifs (-537 to -524 and -339 to -327) for either peroxisome proliferator-activated receptor or farnesoid X-activated receptor are involved in this glucose-mediated transcriptional regulation. This finding suggests that high glucose upregulates the transcription of human PLTP gene via nuclear hormone receptors. In addition, high glucose increases mRNA levels for several genes that are functionally important in HDL metabolism, including human ATP-binding cassette transporter A1, apolipoprotein A-I, scavenger receptor BI, and hepatic lipase. The functional promoter activities of these genes are enhanced by high glucose in three cell lines tested, indicating that glucose may also regulate these genes at the transcriptional level. Our findings provide a molecular basis for a role of hyperglycemia in altered HDL metabolism.

Phospholipid transfer protein (PLTP) plays an important role in plasma lipid and lipoprotein metabolism. We have previously cloned and characterized the promoter region of the human PLTP gene. The present study was conducted to determine if the promoter activity of the human PLTP gene is affected by fibrate, a hypolipidemic drug, and to identify DNA sequences that are responsible for the effect. The results indicated that the promoter activity of the PLTP gene was significantly reduced by fenofibrate, and the area that was mainly responsive to the reducing effect by fibrate was located between -377 and -230 of the 5'-flanking region. The DNA sequence analysis suggested that each area of the DNA sequences from -342 to -323 and from -322 to -299 has two repeated sequences, which are inverted and homologous to the recognition motif of peroxisome proliferator-activated receptor (PPAR), namely the PPAR-responsive element (PPRE). Mutagenesis of these PPRE-like sequences, especially that at -322 to -299, abolished most of the reducing effects of fibrate on the PLTP promoter activity. These findings strongly suggest that the PPRE-like elements are responsible for the reduced promoter activity of the human PLTP gene by fibrate., (Copyright 1999 Academic Press.)

Hypothalamic neuropeptide Y (NPY) and serotonin (5-HT)-containing neurons are believed to exert an interactive effect on ingestive behavior. The present study examined the ability of two serotonergic antagonists, spiperone (SPIP), a 5-HT2A antagonist, and mianserin (MIAN), a 5-HT(2A/2C) antagonist, to block the inhibitory action of the 5-HT(2A/2C) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on NPY-stimulated eating. Drugs were injected directly into the paraventricular nucleus (PVN), the perifornical (PFH) or the ventromedial hypothalamus (VMH) at the onset of the dark cycle. PVN, PFH and VMH injections of NPY potentiated food intake although only PVN pretreatment with DOI (5-20 nmol) suppressed NPY-induced eating. SPIP or MIAN, injected immediately prior to PVN DOI, reversed the suppressive effect of DOI on NPY feeding. These findings are consistent with other recent data showing that 5-HT2A receptors within the PVN modulate NPY's effect on food intake at the start of the nocturnal period.

Because of the paucity of data on phospholipid transfer protein (PLTP) activity and lipoprotein phospholipid in mouse strains, plasma PLTP activity (PLTA), plasma phospholipid and cholesterol, HDL phospholipid and cholesterol, and HDL size distribution were determined in 15 inbred mouse strains. The 15 inbred mouse strains differed in their relatedness to one another and consisted of six largely unrelated groups: Castaneus, Swiss, C57BL, AKR, DBA, and NZB. Lipid and PLTA analyses were performed on plasma pools from male and female mice that had fasted for 4 h prior to blood draw. Among the representative unrelated strains fed the chow diet, there was a highly significant relationship between PLTA and plasma phospholipid (r(s) = 0.727, P < 0.01), HDL phospholipid (r(s) = 0.762, P < 0.01), HDL cholesterol (r(s) = 0.699, P < 0.02), percentage of large HDL particles (r(s) = 0.699, P < 0.02), and HDL peak size (r(s) = 0.776, P < 0.01). Similar results were obtained among these strains fed a high fat, high cholesterol diet. PLTA increased in all strains fed the high fat diet (chix = 94%, range 6 to 221%). Strain SM having relatively low PLTA and HDL was crossed with strain NZB having high PLTA and HDL. The F1 progeny from this cross were backcrossed to strain SM and 41 male backcross progeny collected. Among these individual backcrossed animals, PLTA was highly correlated with plasma phospholipid (r(s) = 0.508, P = 0.001), HDL phospholipid (r(s) = 0.566, P < 0.001), HDL cholesterol (r(s) = 0.532, P < 0.001), and percentage of large HDL particles (r(s) = 0.446, P = 0.020). Therefore, we conclude that PLTP is a determinant of HDL level and size in mice.-Albers, J. J., W. Pitman, G. Wolfbauer, M. C. Cheung, H. Kennedy, A-Y. Tu, S. M. Marcovina, and B. Paigen. Relationship between phospholipid transfer protein activity and HDL level and size among inbred mouse strains.

The human plasma phospholipid transfer protein (PLTP) has been shown to facilitate the transfer of phospholipids between lipoproteins and convert high-density lipoproteins into larger and smaller particles in vitro. To explore the lipid transport function in vivo, transgenic C57BL/6 mice that express the human PLTP gene, driven by its natural promoter, were generated. Little difference in PLTP activity and lipoprotein lipids was observed between transgenic mice and non-transgenic control mice fed the chow diet. In response to an atherogenic high-fat, high-cholesterol, cholic acid containing diet, the PLTP activity increased significantly with time in control mice (62% in males and 34% in females after the high-fat diet for 18 weeks). In contrast, the PLTP activity did not change appreciably in the transgenic mice fed the atherogenic diet. Thus, the introduction of the human transgene suppressed the diet-induced increase in plasma PLTP activity, as evidenced by a decrease in PLTP mRNA in a variety of tissues. High-density lipoprotein levels decreased in mice fed the atherogenic diet, but there was a proportionally greater decrease in transgenic animals than in controls. After 18 weeks on the atherogenic diet, the transgenic animals had high-density lipoprotein-cholesterol and PLTP activity approximately one-half of that of control animals. Non-denaturing gradient gel electrophoresis of plasma indicated that the atherogenic diet decreased the high-density lipoprotein size distribution in control mice. However, high-density lipoprotein particle size distribution of the transgenic mice was shifted to smaller particles compared with control animals (P < 0.001). These findings suggest that PLTP activity can modulate the effects of an atherogenic diet on high-density lipoproteins.

The mouse gene encoding phospholipid transfer protein (PLTP) was cloned from 129/SvJ lambdaFIX(R) II library and characterized for the first time. It is comprised of 16 exons separated by 15 introns. Its gene organization strikingly resembles that encoding the human PLTP; the exon-intron junctions in these two genes are completely conserved. Sequencing analysis reveals that the putative promoter of mouse PLTP gene consists of a TATA-box, a high GC region, and several consensus sequences for the binding of transcription factors. Within the first 200 bp of the 5'-flanking region, the mouse and human PLTP genes share 81.1% identity of nt sequences and contain the consensus sequences for the transcription factors AP-2 and Sp1 at the same locations.

The present study was conducted to determine the essential DNA sequences required for the transcription of the human phospholipid transfer protein gene. Truncation studies revealed that DNA sequences between -230 and -159, particularly those at the upstream region, were responsible for the full promoter activity. This region was able to compete with AP-2 and GRE oligonucleotides for the binding to HepG2 cell nuclear extract as shown by gel mobility shift assay. Further analysis, using site-directed mutagenesis, indicated that DNA sequences identical to Sp1 and highly homologous to GRE and Ap-2 consensus sequences were essential for the transcription. These findings support the concept that several elements, spread over the entire functional promoter, synergistically drive the basal transcription.

Phospholipid transfer protein (PLTP) and lipopolysaccharide-binding protein (LPB) are lipid transfer proteins found in human plasma. PLTP shares 24% sequence similarity with LBP. PLTP mediates the transfer and exchange of phospholipids between lipoprotein particles, whereas LBP transfers bacterial lipopolysaccharide (LPS) either to lipoprotein particles or to CD14, a soluble and cell-surface receptor for LPS. We asked whether PLTP could interact with LPS and mediate the transfer of LPS to lipoproteins or to CD14. PLTP was able to bind and neutralize LPS: incubation of LPS with purified recombinant PLTP (rPLTP) resulted in the inhibition of the ability of LPS to stimulate adhesive responses of neutrophils, and addition of rPLTP to blood inhibited cytokine production in response to LPS. Transfer of LPS by rPLTP was examined using fluorescence dequenching experiments and native gel electrophoresis. The results suggested that rPLTP was able to mediate the exchange of LPS between micelles and the transfer of LPS to reconstituted HDL particles, but it did not transfer LPS to CD14. Consonant with these findings, rPLTP did not mediate CD14-dependent adhesive responses of neutrophils to LPS. These results suggest that while PLTP and LBP both bind and transfer LPS, PLTP is unable to transfer LPS to CD14 and thus does not mediate responses of cells to LPS.

Lipid transfer proteins play an essential role in the intravascular dynamics of lipids among lipoproteins and between lipoproteins and cell membranes. Phospholipid transfer protein has been known for over a decade but, unlike cholesteryl ester transfer protein, has been investigated relatively little with regard to its physiological importance. The recent determination of the phospholipid transfer protein complementary DNA sequence as well as the further characterization of its gene structure will direct future studies toward the understanding of its structure-function correlations, physiological regulation, and clinical assessment at the molecular level. As a member of the lipid-transfer lipopolysaccharide-binding protein gene family, phospholipid transfer protein will attract investigators to studying its possible involvement in lipopolysaccharide or endotoxin interactions in addition to its phospholipid transfer activity.

The role of plasma phospholipid transfer protein (PLTP) in lipoprotein metabolism is poorly understood. In vitro studies suggest that PLTP influences HDL size and composition and transfers phospholipids among lipoproteins. To provide an in vivo model for studies of PLTP physiology, transgenic mice that express human PLTP were generated. Human PLTP transcripts were detected in total RNA from adipose tissue, lung, heart, and spleen of the two distinct lines (A and C) of transgenic mice. Despite minimal expression of human PLTP in the liver of these transgenic mice and similar plasma phospholipid transfer activity in transgenic and non-transgenic mice (19.1 +/- 3.1 vs 18.9 +/- 2.7 mumol/ml/h), differences in lipoprotein levels were observed between transgenic and control mice receiving the same chow diet. Male transgenic mice of line C had significantly higher HDL cholesterol than control mice (76.4 +/- 4.6 vs 71.9 +/- 7.0 mg/dl, p < 0.05) and the male transgenic mice of lines A and C had a significantly lower non-HDL cholesterol (15.1 +/- 4.1 and 15.6 +/- 4.7 vs 20.9 +/- 5.5 mg/dl, P < 0.01 and P < 0.02) and a significantly higher HDL cholesterol/non-HDL cholesterol ratio than the control mice (5.3 +/- 1.3 and 5.5 +/- 2.2 vs 3.9 +/- 1.9 mg/dl, P < 0.01 and P < 0.02). Female mice from transgenic line C had higher HDL cholesterol than control mice (64.6 +/- 4.8 vs 57.4 +/- 5.1 mg/dl, P < 0.01) while female mice from line A tended to have higher HDL cholesterol/non-HDL cholesterol ratio than control mice (5.5 +/- 3.7 vs 3.8 +/- 1.4). These observations suggest that expression of PLTP in peripheral tissues play an important role in lipoprotein metabolism. Expression of human PLTP produced a more favorable lipoprotein profile and thus, enhanced expression of PLTP could potentially retard atherosclerosis.

We have identified the functional promoter region of the human phospholipid transfer protein gene. Primer extension analysis indicates multiple sites for transcription initiation. Sequence analysis reveals that the promoter consists of TATA box, high GC region, and several consensus sequences for the potential binding of transcription factors. To assay promoter activity, DNA fragments with various lengths of the 5'-flanking region were fused upstream to the luciferase gene and transfected into HepG2, COS-7, and CHO cells. A minimal promoter of 159 base pairs between -230 and -72 relative to the first transcriptional initiation site is responsible for the full activity. Two motifs, Sp1 and AP-2, are located within this area. It may suggest that the PLTP promoter activity relies primarily on the putative cis-elements in the functional region.

The molecular cloning of mouse plasma phospholipid transfer protein (PLTP) and the eukaryotic cell expression of complementary DNA for mouse and human PLTP are described. Mouse PLTP was found to share 83% amino acid sequence identity with human PLTP. PLTP was produced in baby hamster kidney cells. Conditioned medium from BHK cells expressing PLTP possessed both phospholipid transfer activity and high density lipoprotein (HDL) conversion activity. PLTP mRNA was detected in all 16 human tissues examined by Northern blot analysis with ovary, thymus, and placenta having the highest levels. PLTP mRNA was also examined in eight mouse tissues with the highest PLTP mRNA levels found in the lung, brain, and heart. The effect of purified human plasma-derived PLTP and human recombinant PLTP (rPLTP) on the two human plasma HDL subspecies Lp(A-I) and Lp(A-I/A-II) was evaluated. Plasma PLTP or rPLTP converted the two distinct size subspecies of Lp(A-I) into a larger species, an intermediate species, and a smaller species. Lp(A-I/A-II) particles containing multiple size subspecies were significantly altered by incubation with either plasma or rPLTP with the largest but less prominent subspecies becoming the predominant one, and the smallest subspecies increasing in concentration. Thus, PLTP promoted the conversion of both Lp(A-I) and Lp(A-I/A-II) to populations of larger and smaller particles. Also, both human PLTP and mouse rPLTP were able to convert human or mouse HDL into larger and smaller particles. These observations suggest that PLTP may play a key role in extracellular phospholipid transport and modulation of HDL particles.

We have determined the exon/intron organization of the human phospholipid transfer protein gene. The gene, which spans approximately 13.3 kilobases, is comprised of 16 exons. The organization of the phospholipid transfer protein gene strikingly resembles that encoding another plasma lipid transfer protein, the human cholesterol ester transfer protein. The exon-intron junctions in these two genes are highly conserved, with eight out of fifteen junctions interrupting the same codons, while the remaining junctions lie within 5 residues of each other. The similarity in gene structure and homology in coding sequences suggests that these two genes most likely evolved from a common ancestral gene.

Phospholipid transfer protein (PLTP) was purified from lipoprotein-free human plasma, obtained upon treatment of plasma with dextran sulfate and Ca2+, by employing a series of column chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified PLTP showed a single main band, corresponding to the molecular mass of 78 kDa. However, isoelectric focusing of the purified preparation gave multiple bands with pI ranging from 4.3 to 5.1, indicative of microheterogeneity. Purified PLTP was shown to possess not only phospholipid transfer activity, but also high density lipoprotein (HDL) conversion activity (Tu, A.-Y., Nishida, H. I., and Nishida, T. (1990), FASEB J. 4, A2148; Jauhiainen, M., Metso, J., Pahlman, R., Blomqvist, S., van Tol, A., and Ehnholm, C. (1993) J. Biol. Chem. 268, 4032-4036). Isolated HDL3 was enlarged to the size of HDL2b upon incubation with purified PLTP for 6 h at 37 degrees C at the PLTP/HDL3 molar ratio of approximately 1:45. Both the HDL conversion and the phosphatidylcholine transfer activities of purified PLTP were effectively inhibited by rabbit anti-PLTP immunoglobulin G. The primary importance of PLTP in the HDL enlargement that occurs in human plasma upon incubation at 37 degrees C was shown by the strong inhibitory effect of the anti-PLTP immunoglobulin G. The process of PLTP-mediated HDL enlargement was accompanied by the release of apoproteins, primarily apoA-I. HDL3 enlargement mediated by PLTP was effectively inhibited by the addition of free fatty acids.