Transglycosylation Activity of Engineered Bifidobacterium Lacto-N-Biosidase Mutants at Donor Subsites for Lacto-N-Tetraose Synthesis

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.     

Methamphetamine Blocks Adenosine A2A Receptor Activation via Sigma 1 and Cannabinoid CB1 Receptors

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 A_2A receptor (A_2AR). First, we noticed that methamphetamine does not affect A_2A functionality if the receptor is expressed in a heterologous system. However, A_2AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB₁ receptor (CB₁R) and the sigma 1 receptor (σ₁R). Signaling via both adenosine A_2AR and cannabinoid CB₁R was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the A_2AR–CB₁R heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the A_2AR- 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, A_2AR, which is a therapeutic target for neuroprotection, and CB₁R, which is the most abundant G protein-coupled receptor (GPCR) in the brain.     

Synthesis of deuterated cyclopropene fatty esters structurally related to palmitic and myristic acids

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.     

Proteomic analysis in cerebrospinal fluid of patients with atypical nonketotic hyperglycinemia and pulmonary hypertension - A pilot study

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.     

Role of Serum Cholesterol and Statin Use in the Risk of Prostate Cancer Detection and Tumor Aggressiveness

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.     

Thiazolium‐Based Catalysts for the Etherification of Benzylic Alcohols under Solvent‐Free Conditions

Thiazolium and imidazolium hybrid materials were prepared by radical reactions between a mercaptopropyl‐modified SBA‐15 mesoporous silica and bis‐vinylthiazolium or bis‐vinylimidazolium dibromide salts. These hybrid materials were characterized by several techniques and were employed in the etherification reaction of 1‐phenylethanol. Solvent‐free conditions at 160 °C under different gas phases (oxygen, air, nitrogen and argon) were used. The thiazolium‐based material displayed excellent performances. Further studies were carried out using unsupported thiazolium salts, with or without a methyl group at the C‐2 position of the thiazolium moiety. These studies allowed us to propose a reaction mechanism. The supported thiazolium‐based material was successfully used in the etherification reaction of two other benzylic alcohols and also in seven consecutive cycles. This work represents the first use of thiazolium‐based compounds as catalysts for the etherification reaction of alcohols.