Frequency decoding of calcium oscillations

Background

Calcium (Ca^2 +) oscillations are ubiquitous signals present in all cells that provide efficient means to transmit intracellular biological information. Either spontaneously or upon receptor ligand binding, the otherwise stable cytosolic Ca^2 + concentration starts to oscillate. The resulting specific oscillatory pattern is interpreted by intracellular downstream effectors that subsequently activate different cellular processes. This signal transduction can occur through frequency modulation (FM) or amplitude modulation (AM), much similar to a radio signal. The decoding of the oscillatory signal is typically performed by enzymes with multiple Ca^2 + binding residues that diversely can regulate its total phosphorylation, thereby activating cellular program. To date, NFAT, NF-κB, CaMKII, MAPK and calpain have been reported to have frequency decoding properties.

Scope of review

The basic principles and recent discoveries reporting frequency decoding of FM Ca^2 + oscillations are reviewed here.

Major conclusions

A limited number of cellular frequency decoding molecules of Ca^2 + oscillations have yet been reported. Interestingly, their responsiveness to Ca^2 + oscillatory frequencies shows little overlap, suggesting their specific roles in cells.

General significance

Frequency modulation of Ca^2 + oscillations provides an efficient means to differentiate biological responses in the cell, both in health and in disease. Thus, it is crucial to identify and characterize all cellular frequency decoding molecules to understand how cells control important cell programs.     

Inhibition of calcium-calmodulin complex formation by vasorelaxant basic dipeptides demonstrated by in vitro and in silico analyses

Background

Tryptophan-histidine (Trp-His) was found to suppress the activity of the Ca^2 +/calmodulin (CaM)-dependent protein kinases II (CaMKII), which requires the Ca^2 +-CaM complex for an initial activation. In this study, we attempted to clarify whether Trp-His inhibits Ca^2 +-CaM complex formation, a CaMKII activator.

Methods

The ability of Trp-His and other peptides to inhibit Ca^2 +-CaM complex formation was investigated by a Ca^2 +-encapsulation fluorescence assay. The peptide-CaM interactions were illustrated by molecular dynamic simulation.

Results

We showed that Trp-His inhibited Ca^2 +-CaM complex formation with a 1:1 binding stoichiometry of the peptide to CaM, considering that Trp-His reduced Hill coefficient of Ca^2 +-CaM binding from 2.81 to 1.92. His-Trp also showed inhibitory activity, whereas Trp + His, 3-methyl His-Trp, and Phe-His did not show significant inhibitory activity, suggesting that the inhibitory activity was due to a peptide skeleton (irrespective of the sequence), a basic amino acid, a His residue, the N hydrogen atom of its imidazole ring, and Trp residue. In silico studies suggested the possibility that Trp-His and His-Trp interacted with the Ca^2 +-binding site of CaM by forming hydrogen bonds with key Ca^2 +-binding residues of CaM, with a binding free energy of − 49.1 and − 68.0 kJ/mol, respectively.

Conclusions

This is the first study demonstrating that the vasoactive dipeptide Trp-His possesses inhibitory activity against Ca^2 +-CaM complex formation, which may elucidate how Trp-His inhibited CaMKII in a previous study.

General significance

The results provide a basic idea that could lead to the development of small peptides binding with high affinity to CaM and inhibiting Ca^2 +-CaM complex formation in the future.     

Sleep deprivation impairs calcium signaling in mouse splenocytes and leads to a decreased immune response

Background

Sleep is a physiological event that directly influences health by affecting the immune system, in which calcium (Ca^2 +) plays a critical signaling role. We performed live cell measurements of cytosolic Ca^2 + mobilization to understand the changes in Ca^2 + signaling that occur in splenic immune cells after various periods of sleep deprivation (SD).

Methods

Adult male mice were subjected to sleep deprivation by platform technique for different periods (from 12 to 72 h) and Ca^2 + intracellular fluctuations were evaluated in splenocytes by confocal microscopy. We also performed spleen cell evaluation by flow cytometry and analyzed intracellular Ca^2 + mobilization in endoplasmic reticulum and mitochondria. Additionally, Ca^2 + channel gene expression was evaluated

Results

Splenocytes showed a progressive loss of intracellular Ca^2 + maintenance from endoplasmic reticulum (ER) stores. Transient Ca^2 + buffering by the mitochondria was further compromised. These findings were confirmed by changes in mitochondrial integrity and in the performance of the store operated calcium entry (SOCE) and stromal interaction molecule 1 (STIM1) Ca^2 + channels.

Conclusions and general significance

These novel data suggest that SD impairs Ca^2 + signaling, most likely as a result of ER stress, leading to an insufficient Ca^2 + supply for signaling events. Our results support the previously described immunosuppressive effects of sleep loss and provide additional information on the cellular and molecular mechanisms involved in sleep function.     

Stimulation of cytosolic and mitochondrial calcium mobilization by indomethacin in Caco-2 cells: Modulation by the polyphenols quercetin,resveratrol and rutin

Background

The effect of indomethacin (INDO) on Ca^2 + mobilization, cytotoxicity, apoptosis and caspase activation and the potential protective effect of quercetin (QUE), resveratrol (RES) and rutin (RUT) were determined in Caco-2 cells.

Methods

Caco-2 cells were incubated with INDO in the presence or absence of QUE, RES or RUT. The concentrations of Ca^2 + in the cytosol (Fluo-3 AM) and mitochondria (Rhod-2 AM) were determined as well as the cytotoxicity (MTT reduction and LDH leakage), apoptosis (TUNEL) and caspase-3 and 9 activities.

Results

INDO promoted Ca^2 + efflux from the endoplasmic reticulum (ER), resulting in an early, but transient, increment of cytosolic Ca^2 + at 3.5 min, followed by a subsequent increment of intra-mitochondrial Ca^2 + at 24 min. INDO also induced cytotoxicity, apoptosis, and increased caspase activities and cytochrome c release. All these alterations were prevented by the inhibitors of the IP3R and RyR receptors, 2-Aminoethoxydiphenyl borate (2-APB) and dantrolene. QUE was the most efficient polyphenol in preventing Ca^2 + mobilization induced by INDO and all of its consequences including cytotoxicity and apoptosis.

Conclusions

In Caco-2 cells, INDO stimulates ER Ca^2 + mobilization, probably through the activation of IP3R and RyR receptors, and the subsequent entry of Ca^2 + into the mitochondria. Polyphenols protected the cells against the Ca^2 + mobilization induced by INDO and its consequences on cytotoxicity and apoptosis.

General significance

These results confirm the possibility of using polyphenols and particularly QUE for the protection of the gastroduodenal mucosa in subjects consuming NSAIDs.     

Aequorin-based genetic approaches to visualize Ca signaling in developing animal systems

Background

In recent years, as our understanding of the various roles played by Ca^2 + signaling in development and differentiation has expanded, the challenge of imaging Ca^2 + dynamics within living cells, tissues, and whole animal systems has been extended to include specific signaling activity in organelles and non-membrane bound sub-cellular domains.

Scope of review

In this review we outline how recent advances in genetics and molecular biology have contributed to improving and developing current bioluminescence-based Ca^2 + imaging techniques. Reporters can now be targeted to specific cell types, or indeed organelles or domains within a particular cell.

Major conclusions

These advances have contributed to our current understanding of the specificity and heterogeneity of developmental Ca^2 + signaling. The improvement in the spatial resolution that results from specifically targeting a Ca^2 + reporter has helped to reveal how a ubiquitous signaling messenger like Ca^2 + can regulate coincidental but different signaling events within an individual cell; a Ca^2 + signaling paradox that until now has been hard to explain.

General significance

Techniques used to target specific reporters via genetic means will have applications beyond those of the Ca^2 + signaling field, and these will, therefore, make a significant contribution in extending our understanding of the signaling networks that regulate animal development. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.     

Fundamental properties of Ca signals

Background

Ca^2 + is a ubiquitous and versatile second messenger that transmits information through changes of the cytosolic Ca^2 + concentration. Recent investigations changed basic ideas on the dynamic character of Ca^2 + signals and challenge traditional ideas on information transmission.

Scope of review

We present recent findings on key characteristics of the cytosolic Ca^2 + dynamics and theoretical concepts that explain the wide range of experimentally observed Ca^2 + signals. Further, we relate properties of the dynamical regulation of the cytosolic Ca^2 + concentration to ideas about information transmission by stochastic signals.

Major conclusions

We demonstrate the importance of the hierarchal arrangement of Ca^2 + release sites on the emergence of cellular Ca^2 + spikes. Stochastic Ca^2 + signals are functionally robust and adaptive to changing environmental conditions. Fluctuations of interspike intervals (ISIs) and the moment relation derived from ISI distributions contain information on the channel cluster open probability and on pathway properties.

General significance

Robust and reliable signal transduction pathways that entail Ca^2 + dynamics are essential for eukaryotic organisms. Moreover, we expect that the design of a stochastic mechanism which provides robustness and adaptivity will be found also in other biological systems. Ca^2 + dynamics demonstrate that the fluctuations of cellular signals contain information on molecular behavior. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

In vivo effects of propyl gallate,a novel Ca sensitizer,in a mouse model of dilated cardiomyopathy caused by cardiac troponin T mutation

Aims

We have previously demonstrated that propyl gallate has a Ca^2 + sensitizing effect on the force generation in membrane-permeabilized (skinned) cardiac muscle fibers. However, in vivo beneficial effects of propyl gallate as a novel Ca^2 + sensitizer remain uncertain. In the present study, we aim to explore in vivo effects of propyl gallate.

Main methods

We compared effects of propyl gallate on ex vivo intact cardiac muscle fibers and in vivo hearts in healthy mice with those of pimobendan, a clinically used Ca^2 + sensitizer. The therapeutic effect of propyl gallate was investigated using a mouse model of dilated cardiomyopathy (DCM) with reduced myofilament Ca^2 + sensitivity due to a deletion mutation ΔK210 in cardiac troponin T.

Key findings

Propyl gallate, as well as pimobendan, showed a positive inotropic effect. Propyl gallate slightly increased the blood pressure without changing the heart rate in healthy mice, whereas pimobendan decreased the blood pressure probably through vasodilation via inhibition of phosphodiesterase and increased the heart rate. Propyl gallate prevented cardiac remodeling and systolic dysfunction and significantly improved the life-expectancy of knock-in mouse model of DCM with reduced myofilament Ca^2 + sensitivity due to a mutation in cardiac troponin T. On the other hand, gallate, a similarly strong antioxidant polyphenol lacking Ca^2 + sensitizing action, had no beneficial effects on the DCM mice.

Significance

These results suggest that propyl gallate might be useful for the treatment of inherited DCM caused by a reduction in the myofilament Ca^2 + sensitivity.     

Inhibition of protein kinase C βII isoform ameliorates methylglyoxal advanced glycation endproduct-induced cardiomyocyte contractile dysfunction

Aims

Accumulation of advanced glycation endproduct (AGE) contributes to diabetic complication including diabetic cardiomyopathy although the precise underlying mechanism still remains elusive. Recent evidence depicted a pivotal role of protein kinase C (PKC) in diabetic complications. To this end, this study was designed to examine if PKCβII contributes to AGE-induced cardiomyocyte contractile and intracellular Ca^2 + aberrations.

Main methods

Adult rat cardiomyocytes were incubated with methylglyoxal-AGE (MG-AGE) in the absence or presence of the PKCβII inhibitor LY333531 for 12 h. Contractile and intracellular Ca^2 + properties were assessed using an IonOptix system including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), rise in intracellular Ca^2 + Fura-2 fluorescence intensity and intracellular Ca^2 + decay. Oxidative stress, O₂⁻ production and mitochondrial integrity were examined using TBARS, fluorescence imaging, aconitase activity and Western blotting.

Key findings

MG-AGE compromised contractile and intracellular Ca^2 + properties including reduced PS, ± dL/dt, prolonged TPS and TR₉₀, decreased electrically stimulated rise in intracellular Ca^2 + and delayed intracellular Ca^2 + clearance, the effects of which were ablated by the PKCβII inhibitor LY333531. Inhibition of PKCβII rescued MG-AGE-induced oxidative stress, O₂⁻ generation, cell death, apoptosis and mitochondrial injury (reduced aconitase activity, UCP-2 and PGC-1α). In vitro studies revealed that PKCβII inhibition-induced beneficial effects were replicated by the NADPH oxidase inhibitor apocynin and were mitigated by the mitochondrial uncoupler FCCP.

Significance

These findings implicated the therapeutic potential of specific inhibition of PKCβII isoform in the management of AGE accumulation-induced myopathic anomalies.     

Induction of TRPV5 expression by small activating RNA targeting gene promoter as a novel approach to regulate cellular calcium transportation

Aim

Promoter-targeted small activating RNAs (saRNAs) have been shown to be able to induce target gene expression, a mechanism known as RNA activation (RNAa). The present study tested whether saRNA can induce the overexpression of TRPV5 in human cells derived from the kidney and subsequently manipulate cell calcium uptake.

Main methods

Three saRNAs complementary to the TRPV5 promoter were synthesized and transfected into cells. TRPV5 expression at the RNA and protein levels was analyzed by quantitative real-time PCR and Western blotting respectively. For functional study, transcellular Ca^2 + transportation was tested by fura-2 analysis. Dihydrotestosterone (DHT), a suppressor of cellular calcium transportation, was administered to challenge the activating effect of selected saRNA.

Key findings

One of these synthesized saRNAs, ds-2939, significantly induced the expression of TRPV5 at both mRNA and protein levels. Fura-2 analysis revealed that the intracellular Ca^2 + concentration was elevated by ds-2939. DHT treatment reduced transmembrane Ca^2 + transport, which was partially antagonized by ds-2939.

Significance

Our results suggest that a saRNA targeting TRPV5 promoter can be utilized to manipulate the transmembrane Ca^2 + transport by upregulating the expression of TRPV5 and may serve as an alternative for the treatment of Ca^2 + balance-related diseases.     

Cholesterol reduction ameliorates glucose-induced calcium handling and insulin secretion in islets from low-density lipoprotein receptor knockout mice

Aims/hypothesis

Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR^−/−) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca^2 + handling in these islets.

Methods

Isolated islets from both LDLR^−/− and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca^2 + level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0–10 mmol/l) of methyl-beta-cyclodextrin (MβCD).

Results

The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR^−/− than in WT islets, paralleled by an impairment of Ca^2 + handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR^−/− compared with WT islets. Removal of excess cholesterol from LDLR^−/− islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca^2 + handling was also normalized in cholesterol-depleted LDLR^−/− islets. Cholesterol removal from WT islets by 0.1 and 1.0 mmol/l MβCD impaired both GSIS and Ca^2 + handling. In addition, at 10 mmol/l MβCD WT islet showed a loss of membrane integrity and higher DNA fragmentation.

Conclusion

Abnormally high (LDLR^−/− islets) or low cholesterol content (WT islets treated with MβCD) alters both GSIS and Ca^2 + handling. Normalization of cholesterol improves Ca^2 + handling and insulin secretion in LDLR^−/− islets.     

GSPE is superior to NAC in the prevention of contrast-induced nephropathy: Might this superiority be related to caspase 1 and calpain 1?

Aims

Our study was intended to evaluate the role of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), caspases 1 and 3 and calpain 1 in the pathogenesis of contrast-induced nephropathy (CIN) and to compare the protective effects of N acetyl cysteine (NAC) and grape seed proanthocyanidin extract (GSPE) against the development of CIN.

Main methods

32 rats were divided into four groups; control, contrast media (CM), CM + NAC and CM + GSPE. CIN was induced by administration of 7 ml/kg diatrizoate. The experiment was discontinued on the ninth day. Blood was collected for blood urea nitrogen (BUN) and creatinine measurement. Rat kidney tissues were removed for histopathological evaluation and the investigation of caspases 1 and 3, iNOS, eNOS, TUNEL and calpain 1.

Key findings

A significant increase in BUN, creatinine, renal histopathological injury, TUNEL, caspases 1, 3, calpain 1, iNOS and eNOS was observed in the CM group compared to the control group. There was amelioration in all these parameters in the CM + GSPE group, while there was no significant amelioration in BUN, creatinine and renal histopathological injury in the CM + NAC group. In addition, calpain 1 staining and creatinine were significantly lower in the CM + GSPE group compared to the CM + NAC group.

Significance

Our study showed, for the first time in the literature, that GSPE has a greater renoprotective effect compared with NAC and that this effective protection may be related to decrease in calpain 1 levels.     

Fluvoxamine rescues mitochondrial Ca transport and ATP production through σ1-receptor in hypertrophic cardiomyocytes

Aims

We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ₁-receptor (σ₁R), ameliorates cardiac hypertrophy and dysfunction via σ₁R stimulation. Although σ₁R on non-cardiomyocytes interacts with the IP₃ receptor (IP₃R) to promote mitochondrial Ca^2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.

Main methods

Here we performed Ca^2 + imaging and measured ATP production to define the role of σ₁Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca^2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.

Key finding

These cardiomyocytes exhibited imbalances in expression levels of σ₁R and IP₃R and impairments in both phenylephrine-induced mitochondrial Ca^2 + mobilization from the SR and ATP production. Interestingly, σ₁R stimulation with fluvoxamine rescued impaired mitochondrial Ca^2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ₁R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ₁Rs suppressed intracellular Ca^2 + mobilization through IP₃Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.

Significance

These results suggest that σ₁R stimulation with fluvoxamine promotes SR-mitochondrial Ca^2 + transport and mitochondrial ATP production, whereas σ₁R stimulation suppresses intracellular Ca^2 + overload through IP₃Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ₁R agonists including fluvoxamine.     

Analysis of IP3 receptors in and out of cells

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP₃R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca^2 + channels.

Scope of the review

In this brief review, we first consider a variety of complementary methods that allow the links between IP₃ binding and channel gating to be defined. How does IP₃ binding to the IP₃-binding core in each IP₃R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP₃, Ca^2 + signals and IP₃R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP₃R signalling.

Major conclusions

All IP₃R are regulated by both IP₃ and Ca^2 +. This allows them to initiate and regeneratively propagate intracellular Ca^2 + signals. The elementary Ca^2 + release events evoked by IP₃ in intact cells are mediated by very small numbers of active IP₃R and the Ca^2 +-mediated interactions between them. The spatial organization of these Ca^2 + signals and their stochastic dependence on so few IP₃Rs highlight the need for methods that allow the spatial organization of IP₃R signalling to be addressed with single-molecule resolution.

General significance

A variety of complementary methods provide insight into the structural basis of IP₃R activation and the contributions of IP₃-evoked Ca^2 + signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Zn rather than Ca or Mg used as a cofactor in non-muscular actin from the oyster to control protein polymerization

Background

The major cytoskeletal protein of most cells is actin, which polymerizes to form actin filaments (F-actin). Each actin monomer (G-actin) contains a divalent alkaline earth metal ion (in vivo Mg^2 +; in vitro usually Ca^2 +) as a cofactor that is crucial for protein polymerization. Prior to this study, however, whether or not other types of metal ions can play the same role as Mg^2 + or Ca^2 + in actins remains unknown.

Methods

A new actin from the gills of oyster (AGO) was prepared and characterized by protein purification techniques, SDS- and native-PAGE, and LC–MS\MS for the first time. The property of this protein was studied by CD, fluorescence and UV/vis spectroscopy, laser light scattering, and TEM.

Results

AGO is a monomer with a MW of ~ 42 kDa. AGO is unique among all known actins in that Zn^2 + is only a naturally binding metal in the protein, and that one native AGO molecule binds 8 zinc ions, which can be removed by EDTA treatment at pH 7.2. The presence of zinc has a great effect on the secondary and tertiary structure of the protein. Correlated with such effect is that these zinc ions in native AGO facilitate protein polymerization, whereas removal of zinc ions from native AGO results in a loss of such polymerization property.

Conclusions

The present work demonstrates that AGO is a novel zinc-binding protein with high capacity, and high selectivity.

General significance

This work extends an understanding of the function of zinc and actin.     

Effects of sub-chronic aluminum chloride exposure on rat ovaries

Aims

This experiment investigated the effects of sub-chronic aluminum chloride (AlCl₃) exposure on rat ovaries.

Main methods

Eighty female Wistar (5 weeks old) rats, weighed 110–120 g, were randomly divided into four treatment groups: control group (CG), low-dose group (LG, 64 mg/kg BW AlCl₃), mid-dose group (MG, 128 mg/kg BW AlCl₃) and high-dose group (HG, 256 mg/kg BW AlCl₃). The AlCl₃ was administered in drinking water for 120 days. The ovarian ultrastructure was observed. The activities of acid phosphatase (ACP), alkaline phosphatase (ALP), succinate dehydrogenase (SDH), Na⁺–K⁺-ATPase, Mg^2 +-ATPase and Ca^2 +-ATPase, the contents of Fe, Cu and Zn, and the protein expression of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) in the ovary were determined.

Key findings

The results showed that the structure of the ovary was disrupted, the activities of ALP, ACP, SDH, Na⁺–K⁺-ATPase, Mg^2 +-ATPase and Ca^2 +-ATPase, the contents of Zn, Fe and the protein expression of FSHR and LHR were lowered, and the content of Cu was increased in AlCl₃-treated rats than those in control.

Significance

The results indicate that sub-chronic AlCl₃ exposure caused the damage of the ovarian structure, the disturbed metabolism of Fe, Zn and Cu and the decreased activities of Na⁺–K⁺-ATPase, Mg^2 +-ATPase and Ca^2 +-ATPase in the ovary, which could result in suppressed energy supply in the ovary. A combination of suppression of energy supply and reduction of expression of FSHR and LHR could inhibit ovulation and corpus luteum development, leading to infertility in female rats.     

Effects of Krill-derived phospholipid-enriched n − 3 fatty acids on Ca regulation system in cerebral arteries from ovariectomized rats

Aims

To investigate the effects of n − 3 polyunsaturated fatty acids on cerebral circulation, ovariectomized (OVX) rats were administered with phospholipids in krill oil (KPL) or triglycerides in fish oil (FTG); effects on the Ca^2 + regulating system in their basilar artery (BA) were then analyzed.

Main methods

The rats were divided into 4 groups: control, OVX, OVX given KPL (OVXP), and OVX given FTG (OVXT) orally, daily for 2 weeks. Time dependent relaxation (TDR) of contractile response to 5HT in BA was determined myographically, Na⁺/Ca^2 + exchanger (NCX) 1 mRNA expression was determined by real time PCR, and nucleotides were analyzed by HPLC.

Key findings

The level of TDR in OVX that was significantly lower in the control was inhibited by l-NAME and indomethacin; TEA inhibited TDR totally in the control but only partly in OVXP and OVXT. Relaxation induced by the addition of 5 mM KCl to the BA pre-contracted with 5-HT was inhibited by TEA in the controls, OVXP and OVXT, but not in OVX. Overexpression of NCX1 mRNA in the BA from OVX was significantly inhibited by FTG. The ratio of ADP/ATP in cerebral arteries from OVX was significantly inhibited by KPL and FTG. Levels of triglyceride and arachidonic acid in the plasma of OVX increased, but were significantly inhibited by KPL and FTG.

Significance

Ovarian dysfunction affects Ca^2 + activated-, ATP-sensitive-K⁺ channels and NCX1, which play crucial roles in the autoregulation of cerebral blood flow. Also, KPL may become as good a supplement as FTG for postmenopausal women.     

Systemic lupus erythematosus: Molecular cloning of fourteen recombinant DNase monoclonal kappa light chains with different catalytic properties

Background

DNase antibodies can play an important role in the pathogenesis of different autoimmune pathologies.

Methods

An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with systemic lupus erythematosus (SLE) was used. The small pools of phage particles displaying DNA binding light chains with different for DNA were isolated by affinity chromatography on DNA-cellulose and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Forty-five of 451 individual colonies were randomly chosen for a study of MLChs with DNase activity. The clones were expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography followed by gel filtration, and studied in detail.

Results

Fifteen of 45 MLChs efficiently hydrolyzed DNA, and fourteen of them demonstrated various optimal concentrations of KCl or NaCl in a 1–100 mM range and showed one or two pH optima in a 4.8–9.1 range. All MLChs were dependent on divalent metal cations: the ratio of relative DNase activity in the presence of Mn^2 +, Ca^2 +, Mg^2 +, Ni^2 +, Zn^2 +, Cu^2 +, and Co^2 + was individual for each MLCh preparation. Fourteen MLChs demonstrated a comparable affinity for DNA (260–320 nM), but different k_cat values (0.02–0.7 min^− 1).

Conclusions

These observations suggest an extreme diversity of DNase abzymes from SLE patients.

General significance

SLE light chain repertoire can serve as a source of new types of DNases.     

The role of calpain in an in vivo model of oxidative stress-induced retinal ganglion cell damage

Purpose

In this study, we set out to establish an in vivo animal model of oxidative stress in the retinal ganglion cells (RGCs) and determine whether there is a link between oxidative stress in the RGCs and the activation of calpain, a major part of the apoptotic pathway.

Materials and methods

Oxidative stress was induced in the RGCs of C57BL/6 mice by the intravitreal administration of 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH, 30 mM, 2 μl). Control eyes were injected with 2 μl of vehicle. Surviving Fluorogold (FG)-labeled RGCs were then counted in retinal flat mounts. Double staining with CellROX and Annexin V was performed to investigate the co-localization of free radical generation and apoptosis. An immunoblot assay was used both to indirectly evaluate calpain activation in the AAPH-treated eyes by confirming α-fodrin cleavage, and also to evaluate the effect of SNJ-1945 (a specific calpain inhibitor: 4% w/v, 100 mg/kg, intraperitoneal administration) in these eyes.

Results

Intravitreal administration of AAPH led to a significant decrease in FG-labeled RGCs 7 days after treatment (control: 3806.7 ± 575.2 RGCs/mm², AAPH: 3156.1 ± 371.2 RGCs/mm², P < 0.01). CellROX and Annexin V signals were co-localized in the FG-labeled RGCs 24 h after AAPH injection. An immunoblot assay revealed a cleaved α-fodrin band that increased significantly 24 h after AAPH administration. Intraperitoneally administered SNJ-1945 prevented the cleavage of α-fodrin and had a neuroprotective effect against AAPH-induced RGC death (AAPH: 3354.0 ± 226.9 RGCs/mm², AAPH+SNJ-1945: 3717.1 ± 614.6 RGCs/mm², P < 0.01).

Conclusion

AAPH administration was an effective model of oxidative stress in the RGCs, showing that oxidative stress directly activated the calpain pathway and induced RGC death. Furthermore, inhibition of the calpain pathway protected the RGCs after AAPH administration.     

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.