Roles of mouse sperm‐associated alpha‐L‐fucosidases in fertilization

Sperm‐associated α‐L ‐fucosidases have been implicated in fertilization in many species. Previously, we documented the existence of α‐L ‐fucosidase in mouse cauda epididymal contents, and showed that sperm‐associated α‐L ‐fucosidase is cryptically stored within the acrosome and reappears within the sperm equatorial segment after the acrosome reaction. The enrichment of sperm membrane‐associated α‐L ‐fucosidase within the equatorial segment of acrosome‐reacted cells implicates its roles during fertilization. Here, we document the absence of α‐L ‐fucosidase in mouse oocytes and early embryos, and define roles of sperm associated α‐L ‐fucosidase in fertilization using specific inhibitors and competitors. Mouse sperm were pretreated with deoxyfuconojirimycin (DFJ, an inhibitor of α‐L ‐fucosidase) or with anti‐fucosidase antibody; alternatively, mouse oocytes were pretreated with purified human liver α‐L ‐fucosidase. Five‐millimolar DFJ did not inhibit sperm–zona pellucida (ZP) binding, membrane binding, or fusion and penetration, but anti‐fucosidase antibody and purified human liver α‐L ‐fucosidase significantly decreased the frequency of these events. To evaluate sperm‐associated α‐L ‐fucosidase enzyme activity in post‐fusion events, DFJ‐pretreated sperm were microinjected into oocytes, and 2‐pronuclear (2‐PN) embryos were treated with 5 mM DFJ with no significant effects, suggesting that α‐L ‐fucosidase enzyme activity does not play a role in post‐fusion events and/or early embryo development in mice. The recognition and binding of mouse sperm to the ZP and oolemma involves the glycoprotein structure of α‐L ‐fucosidase, but not its catalytic action. These observations suggest that deficits in fucosidase protein and/or the presence of anti‐fucosidase antibody may be responsible for some types of infertility. Mol. Reprod. Dev. 80: 273–285, 2013. © 2013 Wiley Periodicals, Inc.     

Characterization of a group of pyrroloquinoline quinone‐dependent dehydrogenases that are involved in the conversion of L‐sorbose to 2‐Keto‐L‐gulonic acid in Ketogulonicigenium vulgare WSH‐001

Ketogulonicigenium vulgare WSH‐001 is an industrial strain used for vitamin C production. Based on genome sequencing and pathway analysis of the bacterium, some of its potential pyrroloquinoline quinone (PQQ)‐dependent dehydrogenases were predicted, including KVU_pmdA_0245, KVU_2142, KVU_2159, KVU_1366, KVU_0203, KVU_0095, and KVU_pmdB_0115. BLAST and function domain searches showed that enzymes encoded by these genes may act as putative PQQ‐dependent L ‐sorbose dehydrogenases (SDH) or L ‐sorbosone dehydrogenases (SNDH). To validate whether these dehydrogenases are PQQ‐dependent or not, these seven putative dehyrogenases were overexpressed in Escherichia coli BL21 (DE3) and purified for characterization. Biochemical and kinetic characterization of the purified proteins have led to the identification of seven enzymes that possess the ability to oxidize L ‐sorbose or L ‐sorbosone to varying degrees. In addition, the dehydrogenation of sorbose in K. vulgare is validated to be PQQ dependent, identification of these PQQ‐dependent dehydrogenases expanded the PQQ‐dependent dehydrogenase family. Besides, the optimal combination of enzymes that could more efficiently catalyze the conversion of sorbose to gulonic acid was proposed. These are important in supporting the development of metabolic engineering strategies and engineering of efficient strains for one‐step production of vitamin C in the future. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1398–1404, 2013     

Channel‐like crystal structure of cinchoninium L‐O‐phosphoserine salt dihydrate

Studies on the interactions between L ‐O‐ phosphoserine, as one of the simplest fragments of membrane components, and the Cinchona alkaloid cinchonine, in the crystalline state were performed. Cinchoninium L ‐O‐phosposerine salt dihydrate (PhSerCin) crystallizes in a monoclinic crystal system, space group P2₁, with unit cell parameters: a = 8.45400(10) Å, b = 7.17100(10) Å, c = 20.7760(4) Å, α = 90°, β = 98.7830(10)°, γ = 90°, Z = 2. The asymmetric unit consists of the cinchoninium cation linked by hydrogen bonds to a phosphoserine anion and two water molecules. Intermolecular hydrogen bonds connecting phosphoserine anions via water molecules form chains extended along the b axis. Two such chains symmetrically related by twofold screw axis create a “channel.” On both sides of this channel cinchonine cations are attached by hydrogen bonds in which the atoms N1, O12, and water molecules participate. This arrangement mimics the system of bilayer biological membrane. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Efficient racemization of N‐phenylacetyl‐D‐glufosinate for L‐glufosinate production

Most amino acids contain chiral centres and exist as both D‐enantiomer and L‐enantiomer. The optically pure enantiomer is often more valuable than the racemate. Enzymatic resolution provides an effective strategy to obtain optically pure amino acids but often results in large amounts of unwanted isomer. In this study, optically pure L‐glufosinate (L‐PPT) was obtained by coupling amidase‐mediated hydrolysis of N‐phenylacetyl‐D,L‐glufosinate with racemization of N‐phenylacetyl‐D‐glufosinate (NPDG), which exclusively exhibits effective herbicidal properties compared with its D‐enantiomer. To improve the yield of L‐PPT, the racemization reaction conditions were optimized, and through single‐factor experiments, the optimal reaction temperature, reaction time, and mole ratio of phenylacetic acid to NPDG were determined to be 150°C, 30 minutes, and 1.5, respectively. The response surface methodology was applied to further optimize the racemization conditions, and the final yield of L‐PPT reached 96.13% with optimum reaction temperature of 154°C, reaction time of 23 minutes, and phenylacetic acid/NPDG mole ratio of 1.7, respectively. Moreover, adding a small amount of acetic anhydride further raised the yield of L‐PPT to 97.02%.     

Conformations and properties of the L‐tryptophyl‐containing peptides in solution,depending on the pH—Theoretical study vs. experiments

The conformational preference and electronic properties of three L ‐tryptophyl‐containing dipeptides, i.e., glycyl‐L ‐tryptophane (H‐Gly‐Trp‐OH), L ‐alanyl‐L ‐tryptophane (H‐Ala‐Trp‐OH), and L ‐methionyl‐L ‐tryptophane (L ‐Met‐Trp‐OH) in solution depending on the pH of the media are studied both theoretically and experimentally. The effect of the protonation of the COO^? and deprotonation of the NH as well as the alkaline hydrolysis of the amide fragment in a strong basic media on the electronic spectra are discussed. Ab initio and density functional theory (DFT) methods as well as the time‐dependent DFT (TD‐DFT) method as a function of the basis set are performed with a view to obtain the geometry and electronic properties of all of the species as well as the intermediate, obtained in the alkaline hydrolysis mechanism. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 727–734, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Roles of the three L‐domains in β‐retrovirus budding

Retroviral Gag protein plays a critical role during the late stage of virus budding and possesses a so‐called L‐domain containing PT/SAP, PPxY, YxxL or FPIV motifs that are critical for efficient budding. Mason–Pfizer monkey virus (M‐PMV) contains PSAP, PPPY, and YADL sequences in Gag. This study was performed to investigate the roles of these three L‐domain‐like sequences in virus replication in three different cell lines, 293T, COS‐7 and HeLa cells. It was found that the PPxY motif plays an essential role in progeny virus production as a major L‐domain in all three cell lines. The PSAP sequence was shown to function as an additional L‐domain in HeLa cells and to promote efficient release of M‐PMV; however, this sequence was dispensable for M‐PMV production in 293T and COS‐7 cells, suggesting that the role of the PSAP motif as an L‐domain in M‐PMV budding is cell type‐dependent. Viruses possessing multiple L‐domains appear to change the L‐domain usage to replicate in various cells. On the other hand, the YADL motif was required for M‐PMV production as a transport signal of Gag to the plasma membrane, but not as an L‐domain.     

Intraspecies transmission of L‐type‐like bovine spongiform encephalopathy detected in Japan

It has been assumed that the agent causing BSE in cattle is a uniform strain (classical BSE); however, different neuropathological and molecular phenotypes of BSE (atypical BSE) have been recently reported. We demonstrated the successful transmission of L‐type‐like atypical BSE detected in Japan (BSE/JP24 isolate) to cattle. Based on the incubation period, neuropathological hallmarks, and molecular properties of the abnormal host prion protein, the characteristics of BSE/JP24 prion were apparently distinguishable from the classical BSE prion and closely resemble those of bovine amyloidotic spongiform encephalopathy prion detected in Italy.     

Preparation of Immobilized L‐Prolinamide Via Enzymatic Polymerization of Phenolic L‐Prolinamide and Evaluation of Its Catalytic Performance for Direct Asymmetric Aldol Reaction

Phenolic L‐prolinamide was allowed to participate in enzymatic polymerization with horseradish peroxidase as the catalyst, generating immobilized L‐prolinamide. The catalytic performance of the resultant polymer‐supported L‐prolinamide for direct asymmetric aldol reaction between aromatic aldehyde and cyclohexanone was studied. The results show that as prepared L‐prolinamide can catalyze the aldol reaction at room temperature in the presence of H₂O. Relevant aldol addition products are obtained with good yields (up to 91%), high diastereoselectivities (up to 6:94 dr), and medium enantioselectivities (up to 87% ee). Moreover, the title polymer‐supported catalyst can be recovered and reused for at least five cycles while the activity remains almost unchanged. Chirality 26:209–213, 2014. © 2014 Wiley Periodicals, Inc.     

Reduced L‐arginine transport contributes to the pathogenesis of myocardial ischemia‐reperfusion injury

Myocardial injury due to ischemia‐reperfusion (I‐R) damage remains a major clinical challenge. Its pathogenesis is complex including endothelial dysfunction and heightened oxidative stress although the key driving mechanism remains uncertain. In this study we tested the hypothesis that the I‐R process induces a state of insufficient L ‐arginine availability for NO biosynthesis, and that this is pivotal in the development of myocardial I‐R damage. In neonatal rat ventricular cardiomyocytes (NVCM), hypoxia‐reoxygenation significantly decreased L ‐arginine uptake and NO production (42 ± 2% and 71 ± 4%, respectively, both P < 0.01), maximal after 2 h reoxygenation. In parallel, mitochondrial membrane potential significantly decreased and ROS production increased (both P < 0.01). NVCMs infected with adenovirus expressing the L ‐arginine transporter, CAT1, and NVCMs supplemented with L ‐arginine both exhibited significant (all P < 0.05) improvements in NO generation and mitochondrial membrane potentials, with a concomitant significant fall in ROS production and lactate dehydrogenase release during hypoxia‐reoxygenation. In contrast, L ‐arginine deprived NVCM had significantly worsened responses to hypoxia‐reoxygenation. In isolated perfused mouse hearts, L ‐arginine infusion during reperfusion significantly improved left ventricular function after I‐R. These improved contractile responses were not dependent on coronary flow but were associated with a significant decrease in nitrotyrosine formation and increases in phosphorylation of both Akt and troponin I. Collectively, these data strongly implicate reduced L ‐arginine availability as a key factor in the pathogenesis of I‐R injury. Increasing L ‐arginine availability via increased CAT1 expression or by supplementation improves myocardial responses to I‐R. Restoration of L ‐arginine availability may therefore be a valuable strategy to ameliorate I‐R injury. J. Cell. Biochem. 108: 156–168, 2009. © 2009 Wiley‐Liss, Inc.     

Bioreaction Engineering Leading to Efficient Synthesis of L‐Glyceraldehyd‐3‐Phosphate

Enantiopure L‐glyceraldehyde‐3‐phosphate (L‐GAP) is a useful building block in natural biological and synthetic processes. A biocatalytic process using glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) catalyzed phosphorylation of L‐glyceraldehyde (L‐GA) by ATP is used for the synthesis of L‐GAP. L‐GAP has a half‐life of 6.86 h under reaction conditions. The activity of this enzyme depends on the Mg²⁺ to ATP molar ratio showing maximum activity at the optimum molar ratio of 0.7. A kinetic model is developed and validated showing a 2D correlation of 99.9% between experimental and numerical data matrices. The enzyme exhibits inhibition by ADP, AMP, methylglyoxal and Ca²⁺, but not by L‐GAP and inorganic orthophosphate. Moreover, equal amount of Ca²⁺ exerts a different degree of inhibition relative to the activity without the addition of Ca²⁺ depending on the Mg²⁺ to ATP molar ratio. If the Mg²⁺ to ATP molar ratio is set to be at the optimum value or less, inorganic hexametaphosphate (PPi6) suppresses the enzyme activity; otherwise PPi6 enhances the enzyme activity. Based on reaction engineering parameters such as conversion, selectivity and specific productivity, evaluation of different reactor types reveals that batchwise operation via stirred‐tank reactor is the most efficient process for the synthesis of L‐GAP.     

Cardiac‐specific overexpression of metallothionein attenuates L‐NAME‐induced myocardial contractile anomalies and apoptosis

Hypertension contributes to the high cardiac morbidity and mortality. Although oxidative stress plays an essential role in hypertensive heart diseases, the mechanism remains elusive. Transgenic mice with cardiac overexpression of metallothionein, a heavy metal‐binding scavenger, were challenged with N^G‐nitro‐L‐arginine methyl ester (L‐NAME) for 14 days prior to measurement of myocardial contractile and intracellular Ca²⁺ anomalies as well as cell signalling mechanisms using Western blot and immunofluorescence analysis. L‐NAME challenge elicited hypertension, macrophage infiltration, oxidative stress, inflammation and cardiac dysfunction manifested as increased proinflammatory macrophage marker F4/80, interleukin‐1β (IL‐1β), intracellular production, LV end systolic and diastolic diameters as well as depressed fractional shortening. L‐NAME treatment reduced mitochondrial membrane potential (MMP), impaired cardiomyocyte contractile and intracellular Ca²⁺ properties as evidenced by suppressed peak shortening, maximal velocity of shortening/relengthening, rise in intracellular Ca²⁺, along with elevated baseline and peak intracellular Ca²⁺. These unfavourable mechanical changes and decreased MMP (except blood pressure and macrophage infiltration) were alleviated by overexpression of metallothionein. Furthermore, the apoptosis markers including BAD, Bax, Caspase 9, Caspase 12 and cleaved Caspase 3 were up‐regulated while the anti‐apoptotic marker Bcl‐2 was decreased by L‐NAME treatment. Metallothionein transgene reversed L‐NAME‐induced changes in Bax, Bcl‐2, BAD phosphorylation, Caspase 9, Caspase 12 and cleaved Caspase 3. Our results suggest that metallothionein protects against L‐NAME‐induced myocardial contractile anomalies in part through inhibition of apoptosis.     

A special reactor design for investigations of mixing time effects in a scaled‐down industrial L‐lysine fed‐batch fermentation process

A specially designed model reactor based on a 42‐L laboratory fermentor was equipped with six stirrers (Rushton turbines) and five cylindrical disks. In this model reactor, the mixing time, Θ₉₀, turned out to be 13 times longer compared with the 42‐L standard laboratory fermentor fitted with two Rushton turbines and four wall‐fixed longitudinal baffles. To prove the suitability of the model reactor for scaledown studies of mixing‐time‐dependent processes, parallel exponential fed‐batch cultivations were carried out with the leucine‐auxotrophic strain, Corynebacterium glutamicum DSM 5715, serving as a microbial test system. L‐ Leucine, the process‐limiting substrate, was fed onto the liquid surface of both reactors. Cultivations were conducted using the same inoculum material and equal oxygen supply. The model reactor showed reduced sugar consumption (−14%), reduced ammonium consumption (−19%), and reduced biomass formation (−7%), which resulted in a decrease in L ‐lysine formation (−12%). These findings were reflected in less specific enzyme activity, which was determined for citrate synthase (CS), phosphoenolpyruvate carboxylase (PEP‐C), and aspartate kinase (AK). The reduced specific activity of CS correlated with lower CO₂ evolution (−36%) during cultivation. The model reactor represents a valuable tool to simulate the conditions of poor mixing and inhomogeneous substrate distribution in bioreactors of industrial scale. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 599–606, 1999.     

Evidence that L‐glutamine is better than L‐alanine as gluconeogenic substrate in perfused liver of weaned fasted rats submitted to short‐term insulin‐induced hypoglycaemia

Gluconeogenesis in livers from overnight fasted weaned rats submitted to short‐term insulin‐induced hypoglycaemia (IIH) was investigated. For this purpose, a condition of hyperinsulinemia/hypoglycaemia was obtained with an intraperitoneal (ip) injection of regular insulin (1.0 U kg^?1). Control group (COG group) received ip saline. The studies were performed 30 min after insulin (IIH group) or saline (COG group) injection. The livers from IIH and COG rats were perfused with L‐alanine (5 mM), L‐lactate (2 mM), L‐glutamine (10 mM) or glycerol (2 mM). Hepatic glucose, L‐lactate and pyruvate production from L‐alanine was not affected by IIH. In agreement with this result, the hepatic ability in producing glucose from L‐lactate or glycerol remained unchanged (IIH group vs. COG group). However, livers from IIH rats showed higher glucose production from L‐glutamine than livers from COG rats and, in the IIH rats, the production of glucose from L‐glutamine was higher than that from L‐alanine. The higher glucose production in livers from the IIH group, when compared with the COG group was due to its entrance further on gluconeogenic pathway. Taken together, the results suggest that L‐glutamine is better than L‐alanine, as gluconeogenic substrate in livers of hypoglyceaemic weaned rats. Copyright © 2008 John Wiley & Sons, Ltd.     

Asymmetrically simultaneous synthesis of L‐homophenylalanine and N6‐protected‐2‐oxo‐6‐amino‐hexanoic acid by engineered Escherichia coli aspartate aminotransferase

L ‐Homophenylalanine (L ‐HPA) and N⁶‐protected‐2‐oxo‐6‐amino‐hexanoic acid (N⁶‐protected‐OAHA) can be used as building blocks for the manufacture of angiotensin‐converting enzyme inhibitors. To synthesize L ‐HPA and N⁶‐protected‐OAHA simultaneously from 2‐oxo‐4‐phenylbutanoic acid (OPBA) and N⁶‐protected‐L ‐lysine, several variants of Escherichia coli aspartate aminotransferase (AAT) were developed by site‐directed mutagenesis and their catalytic activities were investigated. Three kinds of N⁶‐protected‐L ‐lysine were tested as potential amino donors for the bioconversion process. AAT variants of R292E/L18H and R292E/L18T exhibited specific activities of 0.70±0.01 U/mg protein and 0.67±0.02 U/mg protein to 2‐amino‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐lysine) and 2‐amino‐6‐(2,2,2‐trifluoro‐acetylamino)‐hexanoic acid, respectively. E. coli cells expressing R292E/L18H variant were able to convert OPBA and BOC‐lysine to L ‐HPA and 2‐oxo‐6‐tert‐butoxycarbonylamino‐hexanoic acid (BOC‐OAHA) with 96.2% yield in 8 h. This is the first report demonstrating a process for the simultaneous production of two useful building blocks, L ‐HPA and BOC‐OAHA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

High‐level conversion of L‐lysine into 5‐aminovalerate that can be used for nylon 6,5 synthesis

L ‐Lysine is a potential feedstock for the production of bio‐based precursors for engineering plastics. In this study, we developed a microbial process for high‐level conversion of L ‐lysine into 5‐aminovalerate (5AVA) that can be used as a monomer in nylon 6,5 synthesis. Recombinant Escherichia coli WL3110 strain expressing Pseudomonas putida delta‐aminovaleramidase (DavA) and lysine 2‐monooxygenase (DavB) was grown to high density in fed‐batch culture and used as a whole cell catalyst. High‐density E. coli WL3110 expressing DavAB, grown to an optical density at 600 nm (OD₆₀₀) of 30, yielded 36.51 g/L 5AVA from 60 g/L L ‐lysine in 24 h. Doubling the cell density of E. coli WL3110 improved the conversion yield to 47.96 g/L 5AVA from 60 g/L of L ‐lysine in 24 h. 5AVA production was further improved by doubling the L ‐lysine concentration from 60 to 120 g/L. The highest 5AVA titer (90.59 g/L; molar yield 0.942) was obtained from 120 g/L L ‐lysine by E. coli WL3110 cells grown to OD₆₀₀ of 60. Finally, nylon 6,5 was synthesized by bulk polymerization of ?‐caprolactam and δ‐valerolactam prepared from microbially synthesized 5AVA. The hybrid system demonstrated here has promising possibilities for application in the development of industrial bio‐nylon production processes.     

Trace analysis of N‐acetyl‐L‐cysteine using luminol–H2O2 chemiluminescence system catalyzed by silver nanoparticles

N‐Acetyl‐L‐cysteine (NAC) can inhibit the luminol–H₂O₂, reaction, which is catalyzed by silver nanoparticles. Based on this phenomenon a new method was developed for NAC determination. Under optimum conditions, a linear relationship between chemiluminescence intensity and NAC concentration was found in the range 0.034–0.98 µg/mL. The detection limit was 0.010 µg/mL (S/N =3), and the relative standard deviation (RSD) was <5% for 0.480 µg/mL NAC (n =5). This simple, sensitive and inexpensive method has been applied to measure the concentration of NAC in pharmaceutical tablets. Copyright © 2014 John Wiley & Sons, Ltd.