Improvement of γ-decalactone production by stimulating the import of ricinoleic acid and suppressing the degradation of γ-decalactone in Saccharomyces cerevisiae

A number of yeast species can transform ricinoleic acid into γ-decalactone, a high-value compound with fruity aroma, through β-oxidation. This study investigated the effect of l-carnitine on γ-decalactone production by Saccharomyces cerevisiae MF013 to increase the β-oxidation rate. Results showed that l-carnitine shortened the biotransformation period by approximately 10?h and increased γ-decalactone production by 19.5%. γ-Caprolactone, γ-octalactone, and γ-dodecalactone were separately added to the medium to prevent γ-decalactone degradation by yeast cells at the end of biotransformation. γ-Octalactone competitively inhibited γ-decalactone from binding to lactonase, resulting in an 11% increase in γ-decalactone production. This research proposed an effective approach to improve the γ-decalactone production rate, shorten the biotransformation period, and suppress the γ-decalactone degradation in S. cerevisiae.     

Mitochondrial import and degradation of amyloid-β peptide

Mitochondrial dysfunctions associated with amyloid-β peptide (Aβ) accumulation in mitochondria have been observed in Alzheimer's disease (AD) patients' brains and in AD mice models. Aβ is produced by sequential action of β- and γ-secretases cleaving the amyloid precursor protein (APP). The γ-secretase complex was found in mitochondria-associated endoplasmic reticulum membranes (MAM) suggesting that this could be a potential site of Aβ production, from which Aβ is further transported into the mitochondria. In vitro, Aβ was shown to be imported into the mitochondria through the translocase of the outer membrane (TOM) complex. The mitochondrial presequence protease (PreP) is responsible for Aβ degradation reducing toxic effects of Aβ on mitochondrial functions. The proteolytic activity of PreP is, however, lower in AD brain temporal lobe mitochondria and in AD transgenic mice models, possibly due to an increased reactive oxygen species (ROS) production. Here, we review the intracellular mechanisms of Aβ production, its mitochondrial import and the intra-mitochondrial degradation. We also discuss the implications of a reduced efficiency of mitochondrial Aβ clearance for AD. Understanding the underlying mechanisms may provide new insights into mitochondria related pathogenesis of AD and development of drug therapy against AD. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Glutaric aciduria type I and methylmalonic aciduria: Simulation of cerebral import and export of accumulating neurotoxic dicarboxylic acids in in vitro models of the blood–brain barrier and the choroid plexus

Intracerebral accumulation of neurotoxic dicarboxylic acids (DCAs) plays an important pathophysiological role in glutaric aciduria type I and methylmalonic aciduria. Therefore, we investigated the transport characteristics of accumulating DCAs – glutaric (GA), 3-hydroxyglutaric (3-OH-GA) and methylmalonic acid (MMA) – across porcine brain capillary endothelial cells (pBCEC) and human choroid plexus epithelial cells (hCPEC) representing in vitro models of the blood–brain barrier (BBB) and the choroid plexus respectively. We identified expression of organic acid transporters 1 (OAT1) and 3 (OAT3) in pBCEC on mRNA and protein level. For DCAs tested, transport from the basolateral to the apical site (i.e. efflux) was higher than influx. Efflux transport of GA, 3-OH-GA, and MMA across pBCEC was Na⁺-dependent, ATP-independent, and was inhibited by the OAT substrates para-aminohippuric acid (PAH), estrone sulfate, and taurocholate, and the OAT inhibitor probenecid. Members of the ATP-binding cassette transporter family or the organic anion transporting polypeptide family, namely MRP2, P-gp, BCRP, and OATP1B3, did not mediate transport of GA, 3-OH-GA or MMA confirming the specificity of efflux transport via OATs. In hCPEC, cellular import of GA was dependent on Na⁺-gradient, inhibited by NaCN, and unaffected by probenecid suggesting a Na⁺-dependent DCA transporter. Specific transport of GA across hCPEC, however, was not found. In conclusion, our results indicate a low but specific efflux transport for GA, 3-OH-GA, and MMA across pBCEC, an in vitro model of the BBB, via OAT1 and OAT3 but not across hCPEC, an in vitro model of the choroid plexus.     

L and D presequence peptides derived from the precursor of F1β subunit of the ATP synthase inhibit mitochondrial protein import by interaction with import machinery

We investigated the effect of L and D enantiomers of a 25-residue peptide derived from the N-terminal region of the presequence of Nicotiana plumbaginifolia F₁ subunit of the ATP synthase, pF₁(1, 25), on import into spinach leaf mitochondria. Three in vitro synthesized precursor proteins using different import pathways were used. Import of the precursor proteins of F₁ subunit of the ATP synthase, pre-F₁, and the alternative oxidase, pre-AOX, required addition of external ATP, whereas the chimeric precursor containing the N-terminal 84 amino acids of the cytochrome b ₂ precursor protein linked to dihydrofolate reductase, pre-b ₂(1, 84)-DHFR was not dependent on ATP. Import of pre-F₁, and pre-AOX was inhibited already at 1 M and 3 M concentration of the L and D enantiomers, whereas inhibition of import of pre-b ₂(1, 84)-DHFR, occurred at concentrations >10 M of both enantiomers. Binding efficiency of the precursor proteins was not affected by addition of the L and D enantiomers. There was no correlation between inhibition of import of pre-F₁ and pre-AOX and dissipation of membrane potential measured as a decrease of Rhodamine 123 fluorescence quenching. The inhibitory effect of the L and D presequence enantiomers on import of pre-F₁ and pre-AOX was concluded to occur within the outer membrane translocase machinery beyond the initial precursor receptor interaction. Furthermore, the fact that the D enantiomer had the same effect as the natural peptide showed that interaction of the presequence with the import machinery was not dependent on chiral properties of the presequence.     

Parallel import and pacemakers: who pays the ferryman?

Fifty years after its introduction in clinical cardiology, artificial pacing for patients with bradyarrhythmias has made a huge leap forward.1 The development from bulky, simple fixed-rate pacemakers to small, complex, multi-programmable devices paralleled the vast technological achievements of the second half of the 20th century. In the late 1990s hope emerged even for patients with severe heart failure, with the introduction of biventricular pacing which resulted in an additional class I indication according to the recent guideline of the European Society of Cardiology.2 Consequently, the number of implantations has steadily increased, resulting in more than 10,000 implantations (both first implants and replacements) in the Netherlands in 2007.     

The distribution of different classes of nuclear localization signals (NLSs) in diverse organisms and the utilization of the minor NLS-binding site inplantnuclear import factor importin-α

The specific recognition between the import receptor importin-α and the nuclear localization signals (NLSs) is crucial to ensure the selective transport of cargoes into the nucleus. NLSs contain 1 or 2 clusters of positively charged amino acids, which usually bind to the major (monopartite NLSs) or both minor and major NLS-binding sites (bipartite NLSs). In our recent study, we determined the structure of importin-α1a from rice (Oryza sativa), and made 2 observations that suggest an increased utilization of the minor NLS-binding site in this protein. First, unlike the mammalian protein, both the major and minor NLS-binding sites are auto-inhibited in the unliganded rice protein. Second, we showed that NLSs of the “plant-specific” class preferentially bind to the minor NLS-binding site of rice importin-α. Here, we show that a distinct group of “minor site-specific” NLSs also bind to the minor site of the rice protein. We further show a greater enrichment of proteins containing these “plant-specific” and “minor site-specific” NLSs in the rice proteome. However, the analysis of the distribution of different classes of NLSs in diverse eukaryotes shows that in all organisms, the minor site-specific NLSs are much less prevalent than the classical monopartite and bipartite NLSs.     

Importin α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme

SUMOylation, reversible attachment of small ubiquitin-related modifier (SUMO), serves to regulate hundreds of proteins. Consistent with predominantly nuclear targets, enzymes required for attachment and removal of SUMO are highly enriched in this compartment. This is true also for the first enzyme of the SUMOylation cascade, the SUMO E1 enzyme heterodimer, Aos1/Uba2 (SAE1/SAE2). This essential enzyme serves to activate SUMO and to transfer it to the E2-conjugating enzyme Ubc9. Although the last 40 amino acids in yeast Uba2 have been implicated in its nuclear localization, little was known about the import pathways of Aos1, Uba2, and/or of the assembled E1 heterodimer. Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells. Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible. These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.     

Nuclear import of exogenous FGF1 requires the ER-protein LRRC59 and the importins Kpnα1 and Kpnβ1

Fibroblast growth factor 1 (FGF1) taken up by cells into endocytic vesicles can be translocated across vesicular membranes into the cytosol and the nucleus where it has a growth regulatory activity. Previously, leucine-rich repeat containing 59 (LRRC59) was identified as an intracellular binding partner of FGF1, but its biological role remained unknown. Here, we show that LRRC59 is strictly required for nuclear import of exogenous FGF1. siRNA-mediated depletion of LRRC59 did not inhibit the translocation of FGF1 into cytosol, but blocked the nuclear import of FGF1. We also found that an nuclear localization sequence (NLS) in FGF1, Ran GTPase, karyopherin-α1 (Kpnα1), and Kpnβ1 were required for nuclear import of FGF1. Nuclear import of exogenous FGF2, which depends on CEP57/Translokin, was independent of LRRC59, but was dependent on Kpnα1 and Kpnβ1, while the nuclear import of FGF1 was independent of CEP57. LRRC59 is a membrane-anchored protein that localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE). We found that LRRC59 possesses NLS-like sequences in its cytosolic part that can mediate nuclear import of soluble LRRC59 variants, and that the localization of LRRC59 to the NE depends on Kpnβ1. We propose that LRRC59 facilitates transport of cytosolic FGF1 through nuclear pores by interaction with Kpns and movement of LRRC59 along the ER and NE membranes.     

Involvement of binding lipoproteins in the absorption and transport of α-tocopherol in the rat

1. Specific lipoproteins binding alpha-tocopherol but not its known metabolites have been isolated and identified from cytosol of rat intestinal mucosa and from serum. 2. A timestudy of the appearance of the orally administered alpha-[(3)H]tocopherol with these lipoproteins indicates that very-low-density lipoprotein of serum acts as a carrier of the vitamin. 3. The involvement of the mucosal lipoprotein in the absorption of the vitamin from the intestine has been inferred from observations on the amounts of alpha-tocopherol in serum of orotic acid-fed rats where release of lipoproteins from the liver to serum is completely inhibited. A considerable decrease in the association of alpha-tocopherol with serum very-low-density lipoprotein under this condition is interpreted to mean that serum lipoproteins are limiting factors for the transport of the vitamin across the intestine and that this is possibly effected by exchange of alpha-tocopherol between serum very-low-density lipoprotein and mucosal lipoprotein.     

Gender differences in parameters of lipid metabolism and of level of antioxidants in groups of juveniles—the Even and the Europeans

There was performed a comparative evaluation of the mean values of lipid metabolism parameters in youths and girls of the indigenous population (the Even), of the coming population of the north of Irkutsk province, and of the province center (the Europeoids). Gender differences in HDLP have been revealed in adolescents of the coming population, in the glutathione status and the content of liposoluble vitamins in the Even and coming adolescents and in retinol concentration in the province center adolescents. Adaptive changes in the ROS system were found in the Even girls (the higher α-tocopherol and GSH concentrations and the GSH/GSSG ratio as well as a decrease of GSSG relative to the Even youths and the coming population girls, an increase of α-tocopherol relatively to the coming population Europeoids) and youth of the coming population (the higher GSH content and the GSH/GSSG parameter relatively to the Even youths).     

Reciprocal regulation of nuclear import of the yeast MutSα DNA mismatch repair proteins Msh2 and Msh6

DNA mismatch recognition is performed in eukaryotes by two heterodimers known as MutSα (Msh2/Msh6) and MutSβ (Msh2/Msh3) that must reside in the nucleus to function. Two putative Msh2 nuclear localization sequences (NLS) were characterized by fusion to green fluorescent protein (GFP) and site-directed mutagenesis in the context of Msh2. One NLS functioned in GFP targeting assays and both acted redundantly within Msh2. We examined nuclear localization of each of the MutS monomers in the presence and absence of their partners. Msh2 translocated to the nucleus in cells lacking Msh3 and Msh6; however, cells lacking Msh6 showed significantly decreased levels of nuclear Msh2. Furthermore, the overall protein levels of Msh2 were significantly diminished in the absence of Msh6, particularly if Msh2 lacked a functional NLS. Msh3 localized in the absence of Msh2, but Msh6 localization depended on Msh2 expressing functional NLSs. Overall, the nuclear levels of Msh2 and Msh6 decline when the other partner is absent. The data suggest a stabilization mechanism to prevent free monomer accumulation in the cytoplasm.     

Effect of α-Ketoisocaproate and Leucine on the in Vivo Oxidation of Glutamate and Glutamine in the Rat Brain

Leucine and -ketoisocaproate (-KIC) were perfused at increasing concentrations into rat brain hippocampus by microdialysis to mimic the conditions of maple syrup urine disease. The effects of elevated leucine or -KIC on the oxidation of L-[U-¹⁴C]glutamate and L-[U-¹⁴C]glutamine in the brain were determined in the non-anesthetized rat. ¹⁴CO₂ generated by the metabolic oxidation of [^l4C]glutamate and [¹⁴C]glutamine in brain was measured following its diffusion into the eluant during the microdialysis. Leucine and -KIC exhibited differential effects on ¹⁴CO₂ generation from radioactive glutamate or glutamine. Infusion of 0.5 mM -KIC increased [^l4C]glutamate oxidation approximately 2-fold; higher concentrations of -KIC did not further stimulate [¹⁴C]glutamate oxidation. The enhanced oxidation of [¹⁴C]glutamate may be attributed to the function of -KIC as a nitrogen acceptor from [¹⁴C]glutamate yielding [¹⁴C]-ketoglutarate, an intermediate of the tricarboxylic acid cycle. [¹⁴C-]glutamine oxidation was not stimulated as much as [¹⁴C-]glutamate oxidation and only increased at 10 mM -KIC reflecting the extra metabolic step required for its oxidative metabolism. In contrast, leucine had no effect on the oxidation of either [¹⁴C]glutamate or [¹⁴C]glutamine. In maple syrup urine disease elevated -KIC may play a significant role in altered energy metabolism in brain while leucine may contribute to clinical manifestations of this disease in other ways.     

Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218–226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I^−/− mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I^−/− × apoE^−/− mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I^−/− mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218–222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218–222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.     

Role of the tof gene in the production and perpetuation of the λdv plasmid

Molecular Genetics and Genomics - A series of λ derivatives carrying tof mutations were tested for their ability to give rise to plasmid λ dv. Phages carrying tof mutations that distorted...     

The effect of the use of copper carbonate and copper nanoparticles in the diet of rats on the level of β-amyloid and acetylcholinesterase in selected organs

BackgroundCopper has an important role in nervous system function, as a cofactor of many enzymes and in the synthesis of neurotransmitters. Both the dose and the chemical form of copper can determine the impact of this element on metabolism, the neurological system and the immune system.AimsThe aim of the study was to determine whether and in what form the addition of copper changes the level of amyloid beta and acetylcholinesterase level in selected rat tissues.MethodsThirty, healthy, male, albino Wistar rats aged 7 weeks were randomly divided into 3 groups. Three experimental treatments were used to evaluate the effects of different levels and sources of Cu (6.5 mg kg of diet) in the diet: Cu₀ – rats fed a diet without Cu supplementation; Cu_salt – rats fed a diet with CuCO₃ (6.5 mg kg of diet) during two months of feeding; Cu_NPs - rats fed a diet with Cu nanoparticles (6.5 mg kg of diet) during two months of feeding. In blood serum and tissue homogenates there rated the indicators proving the potential neurodegenerative effect and epigenetic DNA damage induced by chemical form of copper or lack of additional copper supplementation in diet were determined. There were analysed: level of acetylcholinesterase, β-amyloid, low-density lipoprotein receptor-related protein 1, apyrimidinic endonuclease, thymidine glycosidase, alkylpurine-DNA-N-glycosylase and glycosylated acetylcholinesterase.ResultsIrrespective of the form of copper added, it was found to increase acetylcholinesterase level in the brain, spleen and liver, as well as in the blood plasma of the rats. Copper in the form of CuCO₃ was found to increase acetylcholinesterase level in the kidneys. The addition of both forms of copper caused a marked increase in the plasma concentration of β-amyloid in comparison with the diet with no added Cu. The addition of both forms of copper caused a marked increase in the plasma concentration of β-amyloid in comparison with the diet with no added Cu.ConclusionsA lack of added Cu in the diet of rats reduces the concentration of amyloid-β in the blood, whereas administration of copper, in the form of either CuNPs or CuCO₃, increases the level of this peptide in the blood. The use of copper in the form of CuNPs in the diet of rats does not increase the level of β-amyloid more than the use of the carbonate form of this element. The use of CuNPs or CuCO₃ in the diet of rats increases acetylcholinesterase level in the brain, spleen, liver, and blood. CuNPs in the diet of rats were not found to increase acetylcholinesterase level to a greater extent than Cu⁺² carbonate.     

Polymorphism and Linkage of the αa-Crystallin Gene in t-Haplotypes of the Mouse

Restriction fragment polymorphisms were used to order the alpha A-crystallin locus (Crya-1) relative to other genes in mouse t-chromatin and to investigate the relatedness of alpha-A-crystallin sequences among different t-haplotypes. Analysis of DNA from t-recombinant mice mapped Crya-1 to the K end of the H-2 complex and within the distal inverted region characteristic of t-haplotypes. Hybridization with Crya-1 cDNA revealed three distinct phenotypic groups among the 17 different t-haplotypes studied. A majority (9 of 17) of the t-haplotypes were classified into a novel group (Crya-1t) characterized by restriction fragments apparently unique to t-chromosomes and therefore thought to contain alpha A-crystallin sequences descended from the original t-chromosome. A second group of t-haplotypes had restriction fragment patterns indistinguishable from those observed among many common inbred strains of mice of the Crya-1a type, and a third restriction fragment pattern, observed only in the tw121 haplotype, was indistinguishable from the fragment pattern for C3H/DiSn (Crya-1b) and several other inbred strains of mice. Thus, with respect to sequences around the Crya-1 locus, different t-haplotypes show restriction fragment polymorphisms, some of which are comparable to those found in wild-type chromosomes and provide further evidence for genetic heterogeneity in DNA from the distal region of t-haplotypes.     

‘Arab invasion’ and decline,or the import of European racial thought by Iranian nationalists

Historical research on western racial theories has grown exponentially in the past few years. A number of scholars have also investigated various cases of ethnic prejudice beyond the western pale, for instance against Koreans in Japan or Muslims in India. Yet, little attention has been given to the modalities in which European nineteenth-century racial thought was adopted in other parts of the world. This article attempts to broach a discussion on this under-analysed aspect of race studies by reviewing one case, that of Arab-hatred in modern Iran. I argue that the intense hatred of Arabs in Iranian nationalist texts derives from a racialized vision of Iranian history born in nineteenth-century European texts. Racialized historiography was selectively imported by the ideologues of Iranian nationalism as it allowed them to explain Iran's weakness in terms of a loss of racial purity.