N-acetyl-L-methionine is a superior protectant of human serum albumin against post-translational oxidation as compared to N-acetyl-L-tryptophan

Sodium octanoate and N-acetyl-L-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, as compared with N-AcTrp, N-acetyl-L-methionine (N-AcMet) is superior in protecting albumin exposed to light during storage. Here, we examine, whether N-AcMet also is better than N-AcTrp to protect albumin against oxidation. Recombinant human serum albumin (rHSA) without and with N-AcMet or N-AcTrp was oxidized by using chloramine-T (CT) as a model compound for mimicking oxidative stress. Oxidation of rHSA was examined by determining carbonyl groups and advanced oxidation protein products. Structural changes were studied by native-PAGE, circular dichroism, intrinsic fluorescence and differential scanning calorimetry. The anti-oxidant capacity of CT-treated rHSA was quantified by its ability to scavenge peroxynitrite and the hydroxyl radical. The pharmacokinetics of indocyanine green-labeled albumin preparations was studied in male mice. We found that the number of chemical modifications and the structural changes of rHSA were significantly smaller in the presence of N-AcMet than in the presence of N-AcTrp. The anti-oxidant properties of CT-exposed rHSA were best protected by adding N-AcMet. Finally, N-AcMet is superior in preserving the normal pharmacokinetics of rHSA. Thus, N-AcMet is superior to N-AcTrp in protecting albumin preparations against oxidation. In addition, N-AcMet is probable also useful for protecting other proteins. Therefore, N-AcMet should be useful as a new and effective stabilizer and antioxidant for albumin isolated from blood, rHSA, albumin-fusion proteins and for preparations of rHSA-therapeutic complexes.     

Oral supplementation with a combination of l-citrulline and l-arginine rapidly increases plasma l-arginine concentration and enhances NO bioavailability

Background

Chronic supplementation with l-citrulline plus l-arginine has been shown to exhibit anti-atherosclerotic effects. However, the short-term action of this combination on the nitric oxide (NO)–cGMP pathway remains to be elucidated. The objective of the present study was to investigate the acute effects of a combination of oral l-citrulline and l-arginine on plasma l-arginine and NO levels, as well as on blood circulation.

Methods

Rats or New Zealand white rabbits were treated orally with l-citrulline, or l-arginine, or a combination of each at half dosage. Following supplementation, plasma levels of l-arginine, NO_x, cGMP and changes in blood circulation were determined sequentially.

Results

l-Citrulline plus l-arginine supplementation caused a more rapid increase in plasma l-arginine levels and marked enhancement of NO bioavailability, including plasma cGMP concentrations, than with dosage with the single amino acids. Blood flow in the central ear artery in rabbits was also significantly increased by l-citrulline plus l-arginine administration as compared with the control.

Conclusion

Our data show for the first time that a combination of oral l-citrulline and l-arginine effectively and rapidly augments NO-dependent responses at the acute stage. This approach may have clinical utility for the regulation of cardiovascular function in humans.     

Cytotoxicity and inhibition of platelet aggregation caused by an l-amino acid oxidase from Bothrops leucurus venom

Background

Multifunctional l-amino acid oxidases (LAAOs) occur widely in snake venoms.

Methods

The l-AAO from Bothrops leucurus (Bl-LAAO) venom was purified using a combination of molecular exclusion and ion-exchange chromatographies. We report some biochemical features of Bl-LAAO associated with its effect on platelet function and its cytotoxicity.

Results

Bl-LAAO is a 60 kDa monomeric glycoprotein. Its N-terminal sequence shows high homology to other members of the snake-venom LAAO family. Bl-LAAO catalyzes oxidative deamination of l-amino acids with the generation of H₂O₂. The best substrates were: l-Met, l-Norleu, l-Leu, l-Phe and l-Trp. The effects of snake venom LAAOs in hemostasis, especially their action on platelet function remain largely unknown. Bl-LAAO dose-dependently inhibited platelet aggregation of both human PRP and washed platelets. Moreover, the purified enzyme exhibited a killing effect in vitro against Leishmania sp., promastigotes, with a very low EC₅₀ of 0.07 μM. Furthermore, the cytotoxicity of Bl-LAAO was observed in the stomach cancer MKN-45, adeno carcinoma HUTU, colorectal RKO and human fibroblast LL-24 cell lines. The enzyme released enough H₂O₂ in culture medium to induce apoptosis in cells in a dose- and time-dependent manner. The biological effects were inhibited by catalase.

Conclusion

Bl-LAAO, a major component of B. leucurus venom, is a cytotoxin acting primarily via the generation of high amounts of H₂O₂ which kill the cells.

General significance

These results allow us to consider the use of LAAOs as anticancer agents, as tools in biochemical studies to investigate cellular processes, and to obtain a better understanding of the envenomation mechanism.     

Molecular pathology of MELAS and l-arginine effects

Background

The pathogenic mechanism of stroke-like episodes seen in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) has not been clarified yet. About 80% of MELAS patients have an A3243G mutation in the mitochondrial tRNA^Leu(UUR) gene, which is the base change at position 14 in the consensus structure of tRNA^Leu(UUR) gene.

Scope of review

This review aims to give an overview on the actual knowledge about the pathogenic mechanism of mitochondrial cytopathy at the molecular levels, the possible pathogenic mechanism of mitochondrial angiopathy to cause stroke-like episodes at the clinical and pathophysiological levels, and the proposed site of action of l-arginine therapy on MELAS.

Major conclusions

Molecular pathogenesis is mainly demonstrated using ρ⁰ cybrid system. The mutation creates the protein synthesis defects caused by 1) decreased life span of steady state amount of tRNA^Leu(UUR) molecules; 2) decreased ratio of aminoacyl-tRNA^Leu(UUR) versus uncharged tRNA^Leu(UUR) molecules; 3) the accumulation of aminoacylation with leucine without any misacylation; 4) accumulation of processing intermediates such as RNA 19, 5) wobble modification defects. All of these loss of function abnormalities are created by the threshold effects of cell or organ to the mitochondrial energy requirement when they establish the phenotype. Mitochondrial angiopathy demonstrated by muscle or brain pathology, as SSV (SDH strongly stained vessels), and by vascular physiology using FMD (flow mediated dilation). MELAS patients show decreased capacity of NO dependent vasodilation because of the low plasma levels of l-arginine and/or of respiratory chain dysfunction. Although the underlying mechanisms are not completely understood in stroke-like episodes in MELAS, l-arginine therapy improved endothelial dysfunction.

General significance

Though the molecular pathogenesis of an A3243G or T3271C mutation of mitochondrial tRNA^Leu(UUR) gene has been clarified as a mitochondrial cytopathy, the underlying mechanisms of stroke-like episodes in MELAS are not completely understood. At this point, l-arginine therapy showed promise in treating of the stroke-like episodes in MELAS. This article is part of a Special Issue entitled Biochemistry of Mitochondria.     

Effects of acute and sub-chronic l-dopa therapy on striatal l-dopa methylation and dopamine oxidation in an MPTP mouse model of Parkinsons disease

Aims

The molecular mechanisms for the loss of 3,4-dihydroxyphenylalanine (l-dopa) efficacy during the treatment of Parkinson's disease (PD) are unknown. Modifications related to catecholamine metabolism such as changes in l-dopa and dopamine (DA) metabolism, the modulation of catecholamine enzymes and the production of interfering metabolites are the primary concerns of this study.

Main methods

Normal (saline) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) pre-treated mice were primed with 100 mg/kg of l-dopa twice a day for 14 days, and a matching group remained l-dopa naïve. l-dopa naive and primed mice received a challenge dose of 100 mg/kg of l-dopa and were sacrificed 30 min later. Striatal catecholamine levels and the expression and activity of catechol-O-methyltransferase (COMT) were determined.

Key findings

Normal and MPTP pre-treated animals metabolize l-dopa and DA similarly during l-dopa therapy. Administration of a challenge dose of l-dopa increased l-dopa and DA metabolism in l-dopa naïve animals, and this effect was enhanced in l-dopa primed mice. The levels of 3-OMD in MPTP pre-treated animals were almost identical to those in normal mice, which we found are likely due to increased COMT activity in MPTP pre-treated mice.

Significance

The results of this comparative study provide evidence that sub-chronic administration of l-dopa decreases the ability of the striatum to accumulate l-dopa and DA, due to increased metabolism via methylation and oxidation. This data supports evidence for the metabolic adaptation of the catecholamine pathway during long-term treatment with l-dopa, which may explain the causes for the loss of l-dopa efficacy.     

In vivo functional brain imaging and a therapeutic trial of l-arginine in MELAS patients

Background

Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is the most common type of mitochondrial disease and is characterized by stroke-like episodes (SEs), myopathy, lactic acidosis, diabetes mellitus, hearing-loss and cardiomyopathy. The causal hypotheses for SEs in MELAS presented to date are angiopathy, cytopathy and neuronal hyperexcitability. L-arginine (Arg) has been applied for the therapy in MELAS patients.

Scope of review

We will introduce novel in vivo functional brain imaging techniques such as MRI and PET, and discuss the pathogenesis of SEs in MELAS patients. We will further describe here our clinical experience with L-arg therapy and discuss the dual pharmaceutical effects of this drug on MELAS.

Major conclusions

Administration of L-arg to MELAS patients has been successful in reducing neurological symptoms due to acute strokes and preventing recurrences of SEs in the chronic phase. L-Arg has dual pharmaceutical effects on both angiopathy and cytopathy in MELAS.

General significance

In vivo functional brain imaging promotes a better understanding of the pathogenesis and potential therapies for MELAS patients. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.     

Neuroprotection by Kukoamine A against oxidative stress may involve N-methyl-d-aspartate receptors

Background

Accumulative evidences have indicated that oxidative-stress and over-activation of N-methyl-d-aspartate receptors (NMDARs) are important mechanisms of brain injury. This study investigated the neuroprotection of Kukoamine A (KuA) and its potential mechanisms.

Methods

Molecular docking was used to discover KuA that might have the ability of blocking NMDARs. Furthermore, the MTT assay, the measurement of LDH, SOD and MDA, the flow cytometry for ROS, MMP and Annexin V-PI double staining, the laser confocal microscopy for intracellular Ca^2 + and western-blot analysis were employed to evaluate the neuroprotection of KuA.

Results

KuA attenuated H₂O₂-induced cell apoptosis, LDH release, ROS production, MDA level, MMP loss, and intracellular Ca^2 + overload (both induced by H₂O₂ and NMDA), as well as increased the SOD activity. In addition, it could modulate the apoptosis-related proteins (Bax, Bcl-2, p53, procaspase-3 and procaspase-9), the SAPKs (ERK, p38), AKT, CREB, NR2A and NR2B expression.

Conclusions

All the results indicated that KuA has the ability of anti-oxidative stress and this effect may partly via blocking NMDARs in SH-SY5Y cells.General significance: KuA might have the potential therapeutic interventions for brain injury.     

Human serum albumin from recombinant DNA technology: Challenges and strategies

Background

As the most abundant protein in the blood, human serum albumin (HSA) plays an important role in maintaining plasma oncotic pressure and fluid balance between the body's compartments. HSA is thus widely used in the clinic to treat diseases. However, the shortage of and safety issues arising from using plasma HSA (pHSA) underscore the importance of recombinant HSA (rHSA) as a promising substitute for pHSA.

Scope of review

Here, we review the production of rHSA, from expression to downstream processing, and highlight the scalability and cost-effectiveness of the two main expression platforms. We also discuss the biosafety of commercially available pharmaceutical rHSA with respect to impurities and contaminants, followed by an analysis of recent progress in preclinical and clinical trials. We emphasise the challenges of producing pharmaceutical-grade rHSA.

Major conclusions

rHSA can be highly expressed in various hosts and seems to be identical to pHSA. rHSA generated from yeast appears to be as efficient and safe as pHSA in a series of preclinical and clinical trials, whereas rHSA from rice seeds exhibits great potential for more cost-effective production. Cost-effective products with no adverse effects will likely play a vital role in future human therapeutics.

General significance

Our understanding of pharmaceutical-grade rHSA production has improved with respect to expression hosts, biochemical properties, downstream processing, and the detection and removal of impurities. However, due to the large dosages required for clinical applications, the production of sufficient quantities of rHSA still presents challenges. This article is part of a Special Issue entitled Serum Albumin.     

Berberine combined with 2-deoxy-d-glucose synergistically enhances cancer cell proliferation inhibition via energy depletion and unfolded protein response disruption

Background

Targeting multiple aspects of cellular metabolism, such as both aerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), has the potential to improve cancer therapeutics. Berberine (BBR), a widely used traditional Chinese medicine, exerts its antitumor effects by inhibiting OXPHOS. 2-Deoxy-d-glucose (2-DG) targets aerobic glycolysis and demonstrates potential anticancer effects in the clinic. We hypothesized that BBR in combination with 2-DG would be more efficient than either agent alone against cancer cell growth.

Methods

The effects of BBR and 2-DG on cancer cell growth were evaluated using the Sulforhodamine B (SRB) method. Cell death was detected with the PI uptake assay, and Western blot, Q-PCR and luciferase reporter assays were used for signaling pathway detection. An adenovirus system was used for gene overexpression.

Results

BBR combined with 2-DG synergistically enhanced the growth inhibition of cancer cells in vitro. Further mechanistic studies showed that the combination drastically enhanced ATP depletion and strongly disrupted the unfolded protein response (UPR). Overexpressing GRP78 partially prevented the cancer cell inhibition induced by both compounds.

Conclusions

Here, we report for the first time that BBR and 2-DG have a synergistic effect on cancer cell growth inhibition related to ATP energy depletion and disruption of UPR.

General significance

Our results propose the potential use of BBR and 2-DG in combination as an anticancer treatment, reinforcing the hypothesis that targeting both aerobic glycolysis and OXPHOS provides more effective cancer therapy and highlighting the important role of UPR in the process.     

Tyrosine residues play an important role in heme detoxification by serum albumin

Background

Serum albumin binds avidly to heme to form heme–serum albumin complex, also called methemalbumin, and this binding is thought to protect against the potentially toxic effects of heme. However, the mechanism of detoxification has not been fully elucidated.

Methods

SDS-PAGE and Western blot were used to determine the efficiency of methemalbumin on catalyzing protein carbonylation and nitration. HPLC was used to test the formation of heme to protein cross-linked methemalbumin.

Results

The peroxidase activity of heme increased upon human serum albumin (HSA) binding. Methemalbumin showed higher efficiency in catalyzing tyrosine oxidation than free heme in the presence of H₂O₂. Methemalbumin catalyzed self-nitration and significantly promoted the nitration of tyrosine in coexistent protein, but decreased the carbonylation of coexistent protein compared with heme. The heme to protein cross-linked form of methemalbumin suggested that HSA trapped the free radical accompanied by the formation of ferryl heme. When tyrosine residues in HSA were modified by iodination, HSA lost of protection effect on protein carbonylation. The low concentration of glutathione could effectively inhibit tyrosine nitration, but had no effect on protein carbonylation.

Conclusion

HSA protects against the toxic effect of heme by transferring the free radical to tyrosine residues in HSA, therefore protecting surrounding proteins from irreversible oxidation, rather than by direct inhibiting the peroxidase activity. The increased tyrosine radicals can be reduced by endogenic antioxidants such as GSH.

General significance

This investigation indicated the important role of tyrosine residues in heme detoxification by HSA and suggested a possible novel mechanism.     

Stimulation by P2X7 receptors of calcium-dependent production of reactive oxygen species (ROS) in rat submandibular glands

Background

Agonists of P2X₇ receptors increase the production of reactive oxygen species (ROS) in immunocytes. In this work we tested this response and its effect on mitochondrial inner membrane potential (Δψ_m) in exocrine glands.

Methods

The production of ROS by rat submandibular glands was investigated by measuring the oxidation of dichlorodihydrofluorescein (DCFH), a fluorescent probe. The Δψ_m was estimated with tetramethylrhodamine.

Results

Activation of P2X₇ receptors by ATP or Bz-ATP increased the production of ROS. This response was not modified by inhibitors of phospholipase A2 or of various kinases. The effect of ATP was calcium-dependent and was blocked by diphenyliodonium, an inhibitor of flavoproteins. It was not affected by rotenone, an inhibitor of the complex I of the mitochondrial electron transfer chain. Scavengers of ROS had no effect on the dissipation of Δψ_m by ATP.

Conclusions

We conclude that, in rat submandibular glands, P2X₇ receptors stimulate in a calcium-dependent manner an oxidase generating ROS, suggesting the involvement of the dual oxidase Duox2. The production of ROS does not contribute to the depolarization of mitochondria by purinergic agonists.

General significance

Purinergic receptors could be regulators of the bactericidal properties of saliva by promoting both the secretion of peroxidase from acinar cells and by activating Duox2.     

Multiple roles of glucose-6-phosphatases in pathophysiology: State of the art and future trends

Background

The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the hydrolysis of glucose-6-phosphate to glucose and inorganic phosphate. The enzyme is a part of a multicomponent system that includes several integral membrane proteins; the catalytic subunit (G6PC) and transporters for glucose-6-phosphate, inorganic phosphate and glucose. The G6PC gene family presently includes three members, termed as G6PC, G6PC2, and G6PC3. Although the three isoforms show a moderate amino acid sequence homology, their membrane topology and catalytic site are very similar. The isoforms are expressed differently in various tissues. Mutations in all three genes have been reported to be associated with human diseases.

Scope of review

The present review outlines the biochemical features of the G6PC gene family products, the regulation of their expression, their role in the human pathology and the possibilities for pharmacological interventions.

Major conclusions

G6PCs emerge as integrators of extra- and intracellular glucose homeostasis. Beside the well known key role in blood glucose homeostasis, the members of the G6PC family seem to play a role as sensors of intracellular glucose and of intraluminal glucose/glucose-6-phosphate in the endoplasmic reticulum.

General significance

Since mutations in the three G6PC genes can be linked to human pathophysiological conditions, the better understanding of their functioning in connection with genetic alterations, altered expression and tissue distribution has an eminent importance.     

Cytosolic ascorbate peroxidase 1 protects organelles against oxidative stress by wounding- and jasmonate-induced H2O2 in Arabidopsis plants

Background

Reactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production.

Methods

We studied the effect of wounding and MeJA treatment on the levels of H₂O₂ and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1).

Results

The KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H₂O₂ accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H₂O₂ were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment.

General significance

This is the first report demonstrating that H₂O₂-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment.     

Propylisopropylacetic acid (PIA), a constitutional isomer of valproic acid,uncompetitively inhibits arachidonic acid acylation by rat acyl-CoA synthetase 4: A potential drug for bipolar disorder

Background

Mood stabilizers used for treating bipolar disorder (BD) selectively downregulate arachidonic acid (AA) turnover (deacylation–reacylation) in brain phospholipids, when given chronically to rats. In vitro studies suggest that one of these, valproic acid (VPA), which is teratogenic, reduces AA turnover by inhibiting the brain long-chain acyl-CoA synthetase (Acsl)4 mediated acylation of AA to AA-CoA. We tested whether non-teratogenic VPA analogues might also inhibit Acsl4 catalyzed acylation, and thus have a potential anti-BD action.

Methods

Rat Acsl4-flag protein was expressed in Escherichia coli, and the ability of three VPA analogues, propylisopropylacetic acid (PIA), propylisopropylacetamide (PID) and N-methyl-2,2,3,3-tetramethylcyclopropanecarboxamide (MTMCD), and of sodium butyrate, to inhibit conversion of AA to AA-CoA by Acsl4 was quantified using Michaelis–Menten kinetics.

Results

Acsl4-mediated conversion of AA to AA-CoA in vitro was inhibited uncompetitively by PIA, with a K_i of 11.4 mM compared to a published K_i of 25 mM for VPA, while PID, MTMCD and sodium butyrate had no inhibitory effect.

Conclusions

PIA's ability to inhibit conversion of AA to AA-CoA by Acsl4 in vitro suggests that, like VPA, PIA may reduce AA turnover in brain phospholipids in unanesthetized rats, and if so, may be effective as a non-teratogenic mood stabilizer in BD patients.     

Plasmodium falciparum antioxidant protein as a model enzyme for a special class of glutaredoxin/glutathione-dependent peroxiredoxins

Background

Peroxiredoxins are important heterogeneous thiol-dependent hydroperoxidases with a variety of isoforms and enzymatic mechanisms. A special subclass of glutaredoxin/glutathione-dependent peroxiredoxins has been discovered in bacteria and eukaryotes during the last decade, but the exact enzymatic mechanisms of these enzymes remain to be unraveled.

Methods

We performed a comprehensive analysis of the enzyme kinetics and redox states of one of these glutaredoxin/glutathione-dependent peroxiredoxins, the antioxidant protein from the malaria parasite Plasmodium falciparum, using steady-state kinetic measurements, site-directed mutagenesis, redox mobility shift assays, gel filtration, and mass spectrometry.

Results

P. falciparum antioxidant protein requires not only glutaredoxin but also glutathione as a true substrate for the reduction of hydroperoxides. One peroxiredoxin cysteine residue and one glutaredoxin cysteine residue are sufficient for catalysis, however, additional cysteine residues of both proteins result in alternative redox states and conformations in vitro with implications for redox regulation. Our data furthermore point to a glutathione-dependent peroxiredoxin activation and a negative subunit cooperativity.

Conclusions

The investigated glutaredoxin/glutathione/peroxiredoxin system provides numerous new insights into the mechanism and redox regulation of peroxiredoxins.

General significance

As a member of the special subclass of glutaredoxin/glutathione-dependent peroxiredoxins, the P. falciparum antioxidant protein could become a reference protein for peroxiredoxin catalysis and regulation.     

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.     

Deciphering metal-induced oxidative damages on glycated albumin structure and function

Background

Metal ions such as copper or zinc are involved in the development of neurodegenerative pathologies and metabolic diseases such as diabetes mellitus. Albumin structure and functions are impaired following metal- and glucose-mediated oxidative alterations. The aim of this study was to elucidate effects of Cu(II) and Zn(II) ions on glucose-induced modifications in albumin by focusing on glycation, aggregation, oxidation and functional aspects.

Methods

Aggregation and conformational changes in albumin were monitored by spectroscopy, fluorescence and microscopy techniques. Biochemical assays such as carbonyl, thiol groups, albumin-bound Cu, fructosamine and amine group measurements were used. Cellular assays were used to gain functional information concerning antioxidant activity of oxidized albumins.

Results

Both metals promoted inhibition of albumin glycation associated with an enhanced aggregation and oxidation process. Metal ions gave rise to the formation of β-amyloid type aggregates in albumin exhibiting impaired antioxidant properties and toxic activity to murine microglia cells (BV2). The differential efficiency of both metal ions to inhibit albumin glycation, to promote aggregation and to affect cellular physiology is compared.

Conclusions and general significance

Considering the key role of oxidized protein in pathology complications, glycation-mediated and metal ion-induced impairment of albumin properties might be important parameters to be followed and fought.     

Homochiral metal-organic coordination networks from l-typtophan

Three homochiral metal-organic coordination networks [Co₂(l-Trp)₂(Py)₆] · Py · (ClO₄)₂ (1), [Ni(l-Trp)(Py)₃] · H₂O · ClO₄ (2) and [Co₂(l-Trp)(INT)₂(H₂O)₂(ClO₄)] (3), all containing natural amino acid l-HTrp (l-typtophan), were hydrothermally synthesized and structurally characterized. The compounds 1 and 2 crystallize in the orthorhombic space group C222₁, with a = 10.731(2) Å, b = 19.709(4) Å, c = 27.365(6) Å and Z = 4 for 1 and a = 10.710(10) Å, b = 20.088(18) Å, c = 27.63(3) Å and Z = 8 for 2, respectively. The compound 3 has the monoclinic space group P2₁, with a = 8.1934(14) Å, b = 13.209(2) Å, c = 12.464(2) Å, β = 104.107(3)° and Z = 2. Both 1 and 2 consist of 1D helical chains. Compound 3 is composed of 2D networks, which further assemble into a 3D supramolecular structure via weak interlayer interactions. The optically pure amino acid l-HTrp plays an important role leading to homochiral structures reported here.     

In vitro characterization of ligand-induced oligomerization of the S. cerevisiae G-protein coupled receptor,Ste2p

Background

The S. cerevisiae α-factor receptor, Ste2p, is a G-protein coupled receptor that plays key roles in yeast signaling and mating. Oligomerization of Ste2p has previously been shown to be important for intracellular trafficking, receptor processing and endocytosis. However the role of ligand in receptor oligomerization remains enigmatic.

Methods

Using functional recombinant forms of purified Ste2p, atomic force microscopy, dynamic light scattering and chemical crosslinking are applied to investigate the role of ligand in Ste2p oligomerization.

Results

Atomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of α-factor nearly double that of Ste2p alone. This observation is supported by complementary dynamic light scattering measurements which indicate a ligand-induced increase in the polydispersity of the Ste2p hydrodynamic radius. Finally, chemical cross-linking of HEK293 plasma membranes presenting recombinant Ste2p indicates α-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis.

Conclusions

α-factor induces oligomerization of Ste2p in vitro and in membrane.

General significance

These results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo.