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Mireia Castejn-Vilatersana Magda Faijes Antoni Planas 《International journal of molecular sciences》2021,22(6)
The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto-N-biosidase from Bifidobacterium bifidum (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto-N-tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from p-nitrophenyl β-lacto-N-bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10–30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT. 相似文献
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Mireia Casanovas Irene Reyes-Resina Alejandro Lillo Jaume Lillo Raul Lpez-Arnau Jorge Camarasa Elena Escubedo Gemma Navarro Rafael Franco 《International journal of molecular sciences》2021,22(5)
Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect is neurodegeneration leading to a decrease in life expectancy. The aim of this paper was to check whether the drug affects one of the receptors involved in neurodegeneration/neuroprotection events, namely the adenosine A2A receptor (A2AR). First, we noticed that methamphetamine does not affect A2A functionality if the receptor is expressed in a heterologous system. However, A2AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB1 receptor (CB1R) and the sigma 1 receptor (σ1R). Signaling via both adenosine A2AR and cannabinoid CB1R was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the A2AR–CB1R heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the A2AR- and the CB1R-mediated activation of the mitogen activated protein kinase (MAPK) pathway. In conclusion, methamphetamine, with the participation of σ1R, alters the expression and function of two interacting receptors, A2AR, which is a therapeutic target for neuroprotection, and CB1R, which is the most abundant G protein-coupled receptor (GPCR) in the brain. 相似文献
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Laura Gosalbo Mireia Barrot Gemma Fabriás Gemma Arsequell Francisco Camps 《Lipids》1993,28(12):1125-1130
To develop a synthesis of tritiated cyclopropene fatty acids (CPFA), compounds that should prove useful for affinity labeling
of desaturases in insect pheromone biosynthetic studies, a series of novel, selectively deuterated CPFA analogues was prepared
and characterized. In methyl [16-2H]12,13-methylene-12-hexadecenoate, the incorporation of deuterium was achieved by treatment of the corresponding ω-chloro
derivative with sodium borodeuteride in dimethylsulfoxide at 70°C for 24 h (67% yield) following conventional procedures.
Alkylation of the tetrahydropyranyl derivative of 13-tridecynol in the presence of lithium diisopropylamide in tetrahydrofuran
at −20°C with 1-chloro-3-iodopropane in hexamethylphosphoramide, followed by Jones oxidation of the crude product, yielded
16-chloro-12-hexadecynoic acid (54%), which was esterified to the corresponding methyl ester by treatment with potassium carbonate
and methyl iodide in dimethylformamide. Treatment of this acetylenic ester with ethyldiazoacetate in the presence of activated
copper-bronze as catalyst followed by hydrolysis in KOH solution at room temperature yielded 16-chloro-12,13-(carboxymethylene)-12-hexadecenoic
acid. This diacid was treated with excess oxalyl chloride to give the corresponding diacyl chloride, which was decarbonylated
in a diethyl ether solution with zinc chloride, and the cyclopropenium ions thus formed were added at −40°C to a methanolic
sodium hydroxide solution of sodium borohydride to give methyl 16-chloro-12,13-methylene-12-hexadecenoate. Analogous procedures
were followed to prepare methyl [17-2H]10,11-methylene-10-hexadecenoate, methyl [17-2H]11,12-methylene-11-hexadecenoate and methyl [17-2H]12,13-methylene-12-hexadecenoate from the corresponding diacids using sodium borodeuteride in the reduction of the cyclopropenium
ions. Alternatively, methyl [2,2,3,3-2H4]hexadecynoate, prepared by reaction of methyl 2,11-hexadecadiynoate with magnesium in deuterated methanol at room temperature,
was submitted to the above cyclopropenylation and reductive decarbonylation sequence to give methyl [2,2,3,3,17-2H5]-11,12-methylene-11-hexadecenoate. In summary, complementary methods for the selective incorporation of one to five deuterium
atoms into cyclopropene fatty acids, at different sites, in moderate to high yields have been developed. The methods should
easily be applicable to the preparation of the corresponding tritiated analogues. 相似文献
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Rodríguez CE Arranz JA Colomé N Bech-Serra JJ Canals F Del Toro M Riudor E 《Proteomics. Clinical applications》2009,3(12):1430-1439
A variant phenotype of nonketotic hyperglycinemia has been described by our group associated with pulmonary hypertension. The aim of this study is to investigate the cerebrospinal fluid proteomes to get an insight into this neurodegenerative process producing leukoencephalopathy with white matter spongiform degeneration. DIGE and MALDI-TOF-TOF analyses were performed to carry out the proteomic study of four patients against three normal controls and one additional control of a classical nonketotic hyperglycinemia. The differential proteomic analysis showed a displacement of some series of spots toward the acidic side. The shifted proteins showed a high degree of carbonylation and increased methionine sulfoxidation was found in cystatin C and in vitamin-D-binding protein. These findings in addition to the increase of serum malondialdehyde concentration provide evidence of an oxidative stress in the patients under study, which is probably systemic rather than mainly confined to the CNS. The similarities of our findings with those found in other neurodegenerative diseases suggest that oxidative damage is commonly involved in these pathologies. DIGE technology improves the 2-D PAGE differential analysis and it is suitable in proteomic studies with a small number of cases. 相似文献
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Monica Coll Anna Fernandez-Falgueras Anna Iglesias Bernat del Olmo Laia Nogue-Navarro Adria Simon Alexandra Perez Serra Marta Puigmule Laura Lopez Ferran Pico Monica Corona Marta Vallverdu-Prats Coloma Tiron Oscar Campuzano Josep Castella Ramon Brugada Mireia Alcalde 《International journal of molecular sciences》2022,23(20)
Molecular screening for pathogenic mutations in sudden cardiac death (SCD)-related genes is common practice for SCD cases. However, test results may lead to uncertainty because of the identification of variants of unknown significance (VUS) occurring in up to 70% of total identified variants due to a lack of experimental studies. Genetic variants affecting potential splice site variants are among the most difficult to interpret. The aim of this study was to examine rare intronic variants identified in the exonic flanking sequence to meet two main objectives: first, to validate that canonical intronic variants produce aberrant splicing; second, to determine whether rare intronic variants predicted as VUS may affect the splicing product. To achieve these objectives, 28 heart samples of cases of SCD carrying rare intronic variants were studied. Samples were analyzed using 85 SCD genes in custom panel sequencing. Our results showed that rare intronic variants affecting the most canonical splice sites displayed in 100% of cases that they would affect the splicing product, possibly causing aberrant isoforms. However, 25% of these cases (1/4) showed normal splicing, contradicting the in silico results. On the contrary, in silico results predicted an effect in 0% of cases, and experimental results showed >20% (3/14) unpredicted aberrant splicing. Thus, deep intron variants are likely predicted to not have an effect, which, based on our results, might be an underestimation of their effect and, therefore, of their pathogenicity classification and family members’ follow-up. 相似文献
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Marta Vallverdú-Prats Ramon Brugada Mireia Alcalde 《International journal of molecular sciences》2022,23(2)
Arrhythmogenic cardiomyopathy is a heritable heart disease associated with desmosomal mutations, especially premature termination codon (PTC) variants. It is known that PTC triggers the nonsense-mediated decay (NMD) mechanism. It is also accepted that PTC in the last exon escapes NMD; however, the mechanisms involving NMD escaping in 5′-PTC, such as reinitiation of translation, are less known. The main objective of the present study is to evaluate the likelihood that desmosomal genes carrying 5′-PTC will trigger reinitiation. HL1 cell lines were edited by CRISPR/Cas9 to generate isogenic clones carrying 5′-PTC for each of the five desmosomal genes. The genomic context of the ATG in-frame in the 5′ region of desmosomal genes was evaluated by in silico predictions. The expression levels of the edited genes were assessed by Western blot and real-time PCR. Our results indicate that the 5′-PTC in PKP2, DSG2 and DSC2 acts as a null allele with no expression, whereas in the DSP and JUP gene, N-truncated protein is expressed. In concordance with this, the genomic context of the 5′-region of DSP and JUP presents an ATG in-frame with an optimal context for the reinitiation of translation. Thus, 5′-PTC triggers NMD in the PKP2, DSG2* and DSC2 genes, whereas it may escape NMD through the reinitiation of the translation in DSP and JUP genes, with no major effects on ACM-related gene expression. 相似文献
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Juan Morote Anna Celma Jacques Planas José Placer Inés de Torres Mireia Olivan Juan Carles Jaume Reventós Andreas Doll 《International journal of molecular sciences》2014,15(8):13615-13623
The aim of this study was to analyze the relationship between statin use along with serum cholesterol levels and prostate cancer (PCa) detection and aggressiveness. Statin users of three years or more and serum cholesterol levels (SC) were assessed in 2408 men scheduled for prostate biopsy. SC was classified as normal (NSC: <200 mg/dL) or high (HSC: >200 mg/dL). High-grade PCa (HGPCa) was considered if the Gleason score was greater than 7. Statin users comprised 30.9% of those studied. The PCa detection rate was 31.2% of men on statins and 37% of non-statin users (p < 0.006). The PCa detection rate was 26.3% in men with NSC and 40.6% in those with HSC (p < 0.001). In the subset of NSC men, the PCa rate was 26.5% for statin users and 26.2% for non-users (p = 0.939), while in men with HSC, the PCa rate was 36.4% for statin users and 42.0% for non-statin users (p = 0.063). The HGPCa rate was 41.8% for statin users and 32.5% for non-users (p = 0.012). NSC men had a 53.8% rate of HGPCa, while the rate was only 27.6% in HSC men (p < 0.001). NSC men on statins had an HGPCa rate of 70.2%, while non-statin users had a rate of 41.2% (p < 0.001). The HGPCa rate for HSC men on statins was 18.8%, while the rate was 30.0% (p = 0.011) for non-users. Logistic regression analysis suggested that serum cholesterol levels could serve as an independent predictor of PCa risk, OR 1.87 (95% CI 1.56–2.24) and HGPCa risk, OR 0.31 (95% CI 0.23–0.44), while statin usage could not. Statin treatment may prevent PCa detection through serum cholesterol-mediated mechanisms. A disturbing increase in the HGPCa rate was observed in statin users who normalized their serum cholesterol. 相似文献
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