Electron flow into cytochrome c coupled with reactive oxygen species from the electron transport chain converts cytochrome c to a cardiolipin peroxidase: role during ischemia–reperfusion

Background

Cytochrome c (Cyt c) is a mobile component of the electron transport chain (ETC.) which contains a tightly coordinated heme iron. In pathologic settings, a key ligand of the cyt c's heme iron, methionine (Met₈₀), is oxidized allowing cyt c to participate in reactions as a peroxidase with cardiolipin as a target. Myocardial ischemia (ISC) results in ETC. blockade and increased production of reactive oxygen species (ROS). We hypothesized that during ischemia–reperfusion (ISC-REP); ROS generation coupled with electron flow into cyt c would oxidize Met₈₀ and contribute to mitochondrial-mediated ETC. damage.

Methods

Mitochondria were incubated with specific substrates and inhibitors to test the contributions of ROS and electron flow into cyt c. Subsequently, cyt c and cardiolipin were analyzed. To test the pathophysiologic relevance, mouse hearts that underwent ISC-REP were tested for methionine oxidation in cyt c.

Results

The combination of substrate/inhibitor showed that ROS production and electron flux through cyt c are essential for the oxidation of methionine residues that lead to cardiolipin depletion. The content of cyt c methionine oxidation increases following ISC-REP in the intact heart.

Conclusions

Increase in intra-mitochondrial ROS coupled with electron flow into cyt c, oxidizes cyt c followed by depletion of cardiolipin. ISC-REP increases methionine oxidation, supporting that cyt c peroxidase activity can form in the intact heart.

General significance

This study identifies a new site in the ETC. that is damaged during cardiac ISC-REP. Generation of a neoperoxidase activity of cyt c favors the formation of a defective ETC. that activates signaling for cell death.     

A novel herbal treatment reduces depressive-like behaviors and increases BDNF levels in the brain of stressed mice

Aims

Depression is a chronic, recurring and potentially life-threatening illness. Current treatments for depression are characterized by a low success rate and associated with a wide variety of side effects. The aim of the present study was to evaluate the behavioral and biological anti-depressant effects of a novel herbal treatment (NHT), as well as to assess its potential side effects, in comparison to treatment with the selective serotonin reuptake inhibitor escitalopram.

Main methods

Depressive-like behavior was evaluated using the forced swim test (FST) and the tail suspension test (TST). Sexual behavior was evaluated following treatment by measuring latency before first mount and number of total mounts. Brain derived neurotrophic factor (BDNF) levels were evaluated using enzyme-linked immunosorbent assay. Serotonin transporter (SERT) levels in the pre-frontal cortex (PFC) and hypothalamus were evaluated using high affinity binding assay.

Key findings

(1) The NHT reduced depressive-like behavior in the FST and TST; (2) BDNF levels in the PFC of mice treated both with the NHT and escitalopram were increased; (3) SERT levels in the hypothalamus were significantly higher in the NHT group, in comparison to escitalopram and the control groups, and significantly lower in the PFC of the NHT group in comparison to the escitalopram group; and (4) the NHT led to less sexual dysfunction, compared to treatment with escitalopram.

Significance

Our NHT has the potential of being highly efficacious in treating depression in humans, while causing minimal to no influence on sexual function.     

DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein

Background

Thiol-mediated redox regulation of proteins plays a key role in many cellular processes.

Methods

To understand the redox status of cysteinyl thiol groups of the desired proteins, we developed a new maleimide reagent: a maleimide-conjugated single strand DNA, DNA-maleimide (DNA-Mal).

Results

DNA-Mal labelled proteins run as a distinct band on SDS-PAGE, with a discrete 9.32 kDa mobility shift per label regardless of the protein species or electrophoretic conditions.

Conclusions

DNA-Mal labels free thiols like standard maleimide reagents, but possesses practical advantages in titration of the number and relative content of free thiols in a protein.

General significance

The versatility of DNA molecule enhances the application of DNA-Mal in a broader range of cysteine containing proteins.     

Underlying mechanisms mediating the antidepressant effects of estrogens

Background

There is an increased risk for depressive symptoms and affective disorders in individuals who experience drastic drops or fluctuations of gonadal hormones. Moreover, clinical studies indicate that estrogens have the potential to be effective in treating depression.

Scope of the review

Possible underlying mechanisms for the antidepressant activity of estrogens are reviewed and discussed.

Major conclusions

Estrogens exert their antidepressant activity via a multimodal mechanism of action by regulating several pathways and functions associated with antidepressive effects. Estrogens increase serotonergic activity by regulating the synthesis and degradation of serotonin, as well as spontaneous firing of the serotonergic neurons in the raphe nuclei. Both pre- and postsynaptic serotonin receptors are shown to be regulated by estrogens. In addition, estrogens are neurotrophic and promote neuroplasticity and neurogenesis. Similar effects are also observed after treatment with current antidepressant therapies. However in stark contrast to current therapies which must be administered chronically to produce an effect, the responses to estrogens are often observed after a single dose. Many of these estrogenic effects, including antidepressant and anxiolytic responses in behavioral models in rodents, appear to be mediated via the estrogen receptor β subtype.

General significance

The rapid onset of action combined with the multifactorial mechanism of action of estrogens indicates that estrogen treatment could complement currently available therapies for depression. Considering safety aspects, selective estrogen receptor β agonists would probably be the optimal estrogenic therapy.     

Association of three SNPs in interleukin-28B with graft hepatic dysfunction after liver transplantation in Chinese Han population

Background

Recurrent graft infection limited the effect of LT, early recognition and prophylaxis of HBV recurrence are very important, and interleukin 28B (IL‐28B) gene was reported to be associated with HBV infection.

Aims

To explore the association between IL-28B single-nucleotide polymorphisms (SNPs) and graft re-infection after liver transplantation(LT).

Methods

21 recipients with hepatitis B virus(HBV) recurrence and 157 recipients without HBV recurrence were included. We studied three SNPs in the promoter region of IL-28B gene at the positions rs12979860, rs12980275 and rs8099917 by HRM analysis (high-resolution melting curve analysis).

Results

Hepatic allograft dysfunction was more likely to be associated with IL-28B SNPs. However, there was no significant difference in the frequencies of IL-28B gene distribution in recipients with or without HBV recurrence.

Conclusion

IL-28B gene polymorphism may be associated with the prognosis of LT recipients but it needs more experiments.     

Receptor for advanced glycation end-product (RAGE) gene polymorphism 2245G/A is associated with pro-inflammatory,oxidative-glycation markers and sRAGE in diabetic retinopathy

Background

Receptor for advanced glycation end-product (RAGE) gene polymorphism 2245G/A is associated with diabetic retinopathy (DR). However, the mechanism on how it affects the disease development is still unclear.

Aim

This study aims to investigate the relationship between 2245G/A RAGE gene polymorphism and selected pro-inflammatory, oxidative-glycation markers in DR patients.

Methods

A total of 371 unrelated type 2 diabetic patients [200 with retinopathy, 171 without retinopathy (DNR)] and 235 healthy subjects were recruited. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism method followed by DNA sequencing. The nuclear and cytosolic extracts from peripheral blood mononuclear cells were used for nuclear factor kappa B (NF-κB) p65 and superoxide dismutase activity measurement respectively. Plasma was used for glutathione peroxidase activity, advanced oxidation protein product (AOPP), monocyte chemoattractant protein (MCP)-1, pentosidine and soluble RAGE (sRAGE) measurements.

Results

DR patients with 2245GA genotype had significantly elevated levels of activated NF-κB p65, plasma MCP-1, AOPP and pentosidine but lower level of sRAGE when compared to DR patients with wild-type 2245GG.

Conclusion

The RAGE gene polymorphism 2245G/A is associated with pro-inflammatory, oxidative-glycation markers and circulating sRAGE in DR patients. Patients with 2245GA RAGE genotype could aggravate DR possibly via NF-κB mediated inflammatory pathway.     

Design,structural and functional characterization of a Temporin-1b analog active against Gram-negative bacteria

Background

Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs.

Methods

Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB_G6A sequence, which was further optimized by increasing the peptide positive charge (TB_KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR.

Results

TB_KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix.

Conclusion

Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides.

General significance

The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process.     

Novel homozygous p.Y395X mutation in the CYP11B1 gene found in a Vietnamese patient with 11β-hydroxylase deficiency

Context

The deficiency of steroid 11β-hydroxylase is caused by mutations in the CYP11B1 gene and is the second major form of congenital adrenal hyperplasia associated with hypertension.

Objective

The objective of this study was to screen the CYP11B1 gene for mutations in one Vietnamese male suffering from congenital adrenal hyperplasia.

Patient

The patient (46,XY) had congenital adrenal hyperplasia. The clinical manifestations presented precocious puberty, hyper-pigmentation and high blood pressure at 4 years.

Results

The patient was a homozygous carrier of a novel mutation located in exon 7 containing a premature stop codon instead of tyrosine at 395 (p.Y395X).

Conclusion

We have identified a novel mutant of the CYP11B1 gene in one Vietnamese family associated with phenotypes of congenital adrenal hyperplasia. The mutant gene p.Y395X produces a truncated form of the polypeptide and abolishes the enzyme activities, leading to a severe phenotype of congenital adrenal hyperplasia.     

Myocardial interstitial serotonin and its major metabolite, 5-hydroxyindole acetic acid levels determined by microdialysis technique in rat heart

Aims

The aim of this study was to elucidate myocardial interstitial serotonin (5-HT) kinetics in the heart, including 5-HT reuptake and enzymatic degradation to 5-hydroxyindole acetic acid (5-HIAA) via monoamine oxidase (MAO).

Main methods

Using microdialysis technique in anesthetized rats, we simultaneously monitored myocardial interstitial levels of 5-HT and its major metabolite, 5-HIAA, in the left ventricle and examined the effects of local administration of a MAO inhibitor, pargyline, or a 5-HT uptake inhibitor, fluoxetine.

Key findings

Pargyline increased dialysate 5-HT concentration from 1.8 ± 0.3 at baseline to 3.9 ± 0.5 nM but decreased dialysate 5-HIAA concentration from 20.7 ± 1.0 at baseline to 15.8 ± 1.4 nM at 60–80 min of administration. Fluoxetine increased dialysate 5-HT concentration from 1.9 ± 0.4 at baseline to 6.5 ± 0.9 nM at 60–80 min of administration, but did not change dialysate 5-HIAA concentration. Local administration of ADP (100 mM) increased dialysate 5-HT and 5-HIAA concentrations. Pargyline did not affect ADP-induced increase in dialysate 5-HT concentration but suppressed ADP-induced increase in dialysate 5-HIAA concentration during 60 min of ADP administration. Fluoxetine increased dialysate 5-HT concentration at 40–60 min of ADP administration, but did not affect ADP-induced increase in dialysate 5-HIAA concentration.

Significance

Simultaneous monitoring of myocardial interstitial 5-HT and 5-HIAA levels provides valuable information on 5-HT kinetics including reuptake and enzymatic degradation by MAO, which play a role in the regulation of myocardial interstitial 5-HT levels at baseline and when 5-HT levels are elevated.     

G-quadruplex DNA recognition by nucleophosmin: New insights from protein dissection

Background

Nucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region.

Methods

Through a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif.

Results

These results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability.

Conclusions

These findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif.

General observation

This study sheds new light in the molecular mechanism of the complex NPM1/G-quadruplex involved in acute myeloid leukemia (AML) disease.     

BJ-PI2, A non-hemorrhagic metalloproteinase from Bothrops jararaca snake venom

Background

Envenoming by Bothrops jararaca can result in local pain, edema, hemorrhage and necrosis, partially mediated by snake venom metalloproteinases (SVMPs). Here, we describe the characterization of BJ-PI2, a P-I class SVMP from B. jararaca venom, and its local tissue actions.

Methods

BJ-PI2 was purified by a combination of gel filtration, anion-exchange chromatography and reverse phase HPLC, and identified by mass spectrometry. Clotting and fibrin(ogen)olytic activities were assayed using conventional methods. Hemorrhagic activity and changes in vascular permeability were examined in rat dorsal skin. Myonecrosis and inflammatory activity were examined in mouse gastrocnemius muscle.

Results

BJ-PI2 was a 23.08 kDa single-chain polypeptide. Tryptic fragments showed highest homology with SVMP insularinase A from Bothrops insularis, but also with B. jararaca SVMP bothrojaractivase; less similarity was observed with B. jararaca SVMPs BJ-PI and jararafibrases II and IV. BJ-PI2 did not clot fibrinogen or rat citrated plasma but had α- and β-fibrinogenolytic activity (inhibited by EDTA and 1,10-phenanthroline but not by PMSF) and attenuated coagulation after plasma recalcification. BJ-PI2 had fibrinolytic activity. BJ-PI2 increased the vascular permeability of rat dorsal skin (inhibited by 1,10-phenanthroline). BJ-PI2 was not hemorrhagic or myonecrotic but caused migration of inflammatory cells. In contrast, venom was strongly hemorrhagic and myonecrotic but caused less infiltration of inflammatory cells.

Conclusions

BJ-PI2 is a non-hemorrhagic, non-myonecrotic, non-coagulant P-I class SVMP that may enhance vascular permeability and inflammatory cell migration in vivo.

General significance

BJ-PI2 contributes to enhanced vascular permeability and inflammatory cell migration after envenoming, but not to venom-induced hemorrhage and necrosis.     

El déficit de vitamina B12 y ácido fólico en la población mayor de 65 años: estudio descriptivo

Introduction

The aim of this study is to establish the prevalence of vitamin B12 and folic acid deficiency in the population of 65 years and over in semi-urban and rural area, as well as investigating the risk factors, and the determining factors of this deficiency in this population and its relationship with the prevalence of concomitant cardiovascular and cognitive diseases.

Materials and methods

A cross-sectrionall study conducted in the Geriatrics Outpatients in the period between 2008 and 2010. Demographic, clinical and laboratory were collected at 3 different times.A bivariate analysis was performed with lack of vitamin B12 and folic acid, as the outcome variables.

Results

Vitamin B12 defieciency was found in 16.5% of the sample, and no folic acid deficiency. A strong association was found with vitamin B12 deficiency and cardiac and cerebrovascular diseases, vascular risk factors and drugs administered in prevention of cardiac events and ischaemic stroke.

Conclusions

There was a higher prevalence of vitamin B12 deficiency in the elderly population in the catchment area of Medina del Campo compared to that found in the literature, but not so with the isolated deficiency of folic acid.     

The effects of ferulic acid on β-amyloid fibrillar structures investigated through experimental and computational techniques

Background

Current research has indicated that small natural compounds could interfere with β-amyloid fibril growth and have the ability to disassemble preformed folded structures. Ferulic acid (FA), which possesses both hydrophilic and hydrophobic moieties and binds to peptides/proteins, is a potential candidate against amyloidogenesis. The molecular mechanisms connected to this action have not been elucidated in detail yet.

Methods

Here the effects of FA on preformed fibrils are investigated by means of a concerted experimental–computational approach. Spectroscopic techniques, such as FTIR, fluorescence, size exclusion chromatography and confocal microscopy in combination with molecular dynamics simulations are used to identify those features which play a key role in the destabilization of the aggregates.

Results

Experimental findings highlight that FA has disruptive effects on the fibrils. The computational analysis suggests that dissociation of peptides from the amyloid superstructures could take place along the fibril axis and be primarily determined by the cooperative rupture of the backbone hydrogen bonds and of the Asp-Lys salt bridges.

Conclusion

FA clusters could induce a sort of stabilization and tightening of the fibril structure in the short term and its disruption in the long term, inhibiting further fibril re-assembly through FA screening effects.

General significance

The combination of experimental and computational techniques could be successfully used to identify the disrupting action of FA on preformed Aβ fibrils in water solution.     

Purification,characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

Background

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.

Methods

Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.

Results

A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.

Conclusions

The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.

General significance

CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.