A comparison of ceruloplasmin to biological polyanions in promoting the oxidation of Fe under physiologically relevant conditions

Background

Iron oxidation is thought to be predominantly handled enzymatically in the body, to minimize spontaneous combustion with oxygen and to facilitate cellular iron export by loading transferrin. This process may be impaired in disease, and requires more accurate analytical assays to interrogate enzymatic- and auto-oxidation within a physiologically relevant environment.

Method

A new triplex ferroxidase activity assay has been developed that overcomes the previous assay limitations of measuring iron oxidation at a physiologically relevant pH and salinity.

Results

Revised enzymatic kinetics for ceruloplasmin (V_max ≈ 35 μM Fe^3 +/min/μM; K_m ≈ 15 μM) are provided under physiological conditions, and inhibition by sodium azide (K_i for Ferric Gain 78.3 μM, K_i for transferrin loading 8.1 × 10⁴ μM) is quantified. We also used this assay to characterize the non-enzymatic oxidation of iron that proceeded linearly under physiological conditions.

Conclusions and general significance

These findings indicate that the requirement of an enzyme to oxidize iron may only be necessary under conditions of adverse pH or anionic strength, for example from hypoxia. In a normal physiological environment, Fe^3 + incorporation into transferrin would be sufficiently enabled by the biological polyanions that are prevalent within extracellular fluids.     

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.     

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.     

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7

Background

Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (C_P) it lacks the resolving Cys (C_R), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs.

Methods

Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme–substrate and protein–protein interaction were analyzed by molecular docking and surface plasmon resonance analysis.

Results

Oxidation of the C_P is fast (k_+ 1 > 10³ M^− 1 s^− 1), however the rate of reduction by GSH is slow (k′_+ 2 = 12.6 M^− 1 s^− 1) even though molecular docking indicates a strong GSH–GPx7 interaction. Instead, the oxidized C_P can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k_+ 1 > 10³ M^− 1 s^− 1), but not by Trx. By surface plasmon resonance analysis, a K_D = 5.2 μM was calculated for PDI–GPx7 complex. Participation of an alternative non-canonical C_R in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo.

Conclusions

GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates.

General significance

In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.     

Calorimetric analysis of the plasma proteome: Identification of type 1 diabetes patients with early renal function decline

Background

Microalbuminuria (MA) has been questioned as a predictor of progressive renal dysfunction in patients with type 1 diabetes (T1D). Consequently, new clinical end points are needed that identify or predict patients that are at risk for early renal function decline (ERFD). The potential clinical utility of differential scanning calorimetry (DSC) analysis of blood plasma and other biofluids has recently been reported. This method provides an alternate physical basis with which to study disease-associated changes in the bulk plasma proteome.

Methods

DSC analysis of blood plasma was applied to identify unique signatures of ERFD in subjects enrolled in the 1st Joslin Study of the Natural History of Microalbuminuria in Type 1 Diabetes, a prospective cohort study of T1D patients. Recent data suggests that differences in the plasma peptidome of these patients correlate with longitudinal measures of renal function. Differences in DSC profile (thermogram) features were evaluated between T1D MA individuals exhibiting ERFD (n = 15) and matched control subjects (n = 14).

Results

The average control group thermogram resembled a previously defined healthy thermogram. Differences were evident between ERFD and control individuals. Heat capacity values of the main two transitions were found to be significant discriminators of patient status.

Conclusions

Results from this pilot study suggest the potential utility of DSC proteome analysis to prognostic indicators of renal disease in T1D.

General significance

DSC shows sensitivity to changes in the bulk plasma proteome that correlate with clinical status in T1D providing additional support for the utility of DSC profiling in clinical diagnostics.     

Fibronectin stimulates migration through lipid raft dependent NHE-1 activation in mouse embryonic stem cells: Involvement of RhoA,Ca/CaM,and ERK

Background

Extracellular matrix (ECM) components and intracellular pH (pH_i) may serve as regulators of cell migration in various cell types.

Methods

The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na⁺/H⁺ exchanger (NHE)-1 activity was evaluated by measuring pH_i and [²²Na⁺] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.

Results

ECM components (FN, laminin, fibrinogen, and collagen type I) increased [²²Na⁺] uptake, pH_i, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca^2 + influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca^2 +/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [²²Na⁺] uptake and pH_i. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.

Conclusions

FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca^2 +/CaM signaling pathways.

General significance

The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.     

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.     

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.     

Whole blood,flow-chamber studies in real-time indicate a biphasic role for thymosin β-4 in platelet adhesion

Background

Thymosin beta 4 (Tβ₄) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.

Methods

In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ₄, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.

Results

The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s^− 1, corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ₄ (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (> 1 μM) the Tβ₄-potentiating effect was diminished to near control levels. Tβ₄ did interact with fibrinogen with an estimated K_D of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.

Conclusion

These results suggest that Tβ₄ could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ₄ could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.

General significance

This work suggests that Tβ₄ might have a dual role in platelet function.     

Interaction of heparin and heparin-derived oligosaccharides with synthetic peptide analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein

Background

Although protamine is effective as an antidote of heparin, there is a need to replace protamine due to its side effects. HIP peptide has been reported to neutralize the anticoagulant activity of heparin. The interaction of HIP analog peptides with heparin and heparin-derived oligosaccharides is investigated in this paper.

Methods

Seven analogues of the heparin-binding domain of heparin/heparan sulfate-interacting protein (HIP) were synthesized, and their interaction with heparin was characterized by heparin affinity chromatography, isothermal titration calorimetry, and NMR.

Results

NMR results indicate the imidazolium groups of the His side chains of histidine-containing Hip analog peptide interact site-specifically with heparin at pH 5.5. Heparin has identical affinities for HIP analog peptides of opposite chirality. Analysis by counterion condensation theory indicates the peptide AC-SRPKAKAKAKAKDQTK-NH₂ makes on average ∼ 3 ionic interactions with heparin that result in displacement of ∼ 2 Na⁺ ions, and ionic interactions account for ∼ 46% of the binding free energy at a Na⁺ concentration of 0.15 M.

Conclusions

The affinity of heparin for the peptides is strongly dependent on the nature of the cationic side chains and pH. The thermodynamic parameters measured for the interaction of HIP peptide analogs with heparin are strongly dependent on the peptide sequence and pH.

General significance

The information obtained in this research will be of use in the design of new agents for neutralization of the anticoagulant activity of heparin. The site-specific binding of protonated histidine side chains to heparin provides a molecular-level explanation for the pH-dependent binding of β-amyloid peptides by heparin and heparan sulfate proteoglycan and may have implications for amyloid formation.     

Inhibition by polyamines of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid

Background

Viroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication.

Methods

In vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors.

Results

Aminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with K_i of 17 ± 5 μM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with K_i of 32 ± 5 μM and IC₅₀ of 177 ± 5 nM, respectively.

Conclusions

This study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups.

General significance

This fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs.     

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.     

Allosteric modulation of zinc speciation by fatty acids

Background

Serum albumin is the major protein component of blood plasma and is responsible for the circulatory transport of a range of small molecules that include fatty acids, hormones, metal ions and drugs. Studies examining the ligand-binding properties of albumin make up a large proportion of the literature. However, many of these studies do not address the fact that albumin carries multiple ligands (including metal ions) simultaneously in vivo. Thus the binding of a particular ligand may influence both the affinity and dynamics of albumin interactions with another.

Scope of review

Here we review the Zn^2 + and fatty acid transport properties of albumin and highlight an important interplay that exists between them. Also the impact of this dynamic interaction upon the distribution of plasma Zn^2 +, its effect upon cellular Zn^2 + uptake and its importance in the diagnosis of myocardial ischemia are considered.

Major conclusions

We previously identified the major binding site for Zn^2 + on albumin. Furthermore, we revealed that Zn^2 +-binding at this site and fatty acid-binding at the FA2 site are interdependent. This suggests that the binding of fatty acids to albumin may serve as an allosteric switch to modulate Zn^2 +-binding to albumin in blood plasma.

General significance

Fatty acid levels in the blood are dynamic and chronic elevation of plasma fatty acid levels is associated with some metabolic disorders such as cardiovascular disease and diabetes. Since the binding of Zn^2 + to albumin is important for the control of circulatory/cellular Zn^2 + dynamics, this relationship is likely to have important physiological and pathological implications. This article is part of a Special Issue entitled Serum Albumin.     

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.     

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.     

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na/K-ATPase

Background

Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na⁺/K⁺-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na⁺/K⁺-ATPase expression and activity in rats injected with Bothrops alternatus snake venom.

Methods

Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na⁺/K⁺-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively.

Results

Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na⁺/K⁺-ATPase α₁ subunit were increased 6 h post-venom, whereas Na⁺/K⁺-ATPase activity increased 6 h and 24 h post-venom.

Conclusions

Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na⁺/K⁺-ATPase expression and activity in the early phase of renal damage.

General significance

Enhanced Na⁺/K⁺-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.     

The sedimentation properties of ferritins. New insights and analysis of methods of nanoparticle preparation

Background

Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.

Methods

Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.

Results

More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe²⁺ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s_20,w = 17.0 S and D_20,w = 3.21 × 10⁻⁷ cm²/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s_20,w = 18.3 S and D_20,w = 3.18 × 10⁻⁷ cm²/s.

Significance

The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.     

Energetics,conformation, and recognition of DNA duplexes containing a major adduct of an anticancer azolato-bridged dinuclear Pt complex

Background

The design of anticancer metallodrugs is currently focused on platinum complexes which form on DNA major adducts that cannot readily be removed by DNA repair systems. Hence, antitumor azolato-bridged dinuclear Pt^II complexes, such as [{cis-Pt(NH₃)₂}₂(μ‐OH)(μ-pyrazolate)]²⁺ (AMPZ), have been designed and synthesized. These complexes exhibit markedly higher toxic effects in tumor cell lines than mononuclear conventional cisplatin.

Methods

Biophysical and biochemical aspects of the alterations induced in short DNA duplexes uniquely and site-specifically modified by the major DNA adduct of AMPZ, namely 1,2-GG intrastrand cross-links, were examined. Attention was also paid to conformational distortions induced in DNA by the adducts of AMPZ and cisplatin, associated alterations in the thermodynamic stability of the duplexes, and recognition of these adducts by high-mobility-group (HMG) domain proteins.

Results

Chemical probing of DNA conformation, DNA bending studies and translesion synthesis by DNA polymerase across the platinum adduct revealed that the distortion induced in DNA by the major adduct of AMPZ was significantly less pronounced than that induced by similar cross-links from cisplatin. Concomitantly, the cross-link from AMPZ reduced the thermodynamic stability of the modified duplex considerably less. In addition, HMGB1 protein recognizes major DNA adducts of AMPZ markedly less than those of cisplatin.

General significance

The experimental evidence demonstrates why the major DNA adducts of the new anticancer azolato-bridged dinuclear Pt^II complexes are poor substrates for DNA repair observed in a previously published report. The relative resistance to DNA repair explains why these platinum complexes show major pharmacological advantages over cisplatin in tumor cells.     

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.