Acetonation of l-pentoses and 6-deoxy-l-hexoses under kinetic control using heterogeneous acid catalysts

The fibrous polymer-supported sulfonic acid catalyst Smopex-101 H⁺ proved to be an efficient catalyst for the preparation of O-isopropylidene derivatives from a series of rare sugars. Acetonation of the reducing sugars l-arabinose, l-ribose, l-xylose, l-fucose, and l-rhamnose in N,N-dimethylformamide by 2,2-dimethoxypropane or 2-methoxypropene led to the formation of the kinetically favored di-O- and/or mono-O-isopropylidene derivatives in 46-88% yields. The method consists of a simple experimental procedure which does not require predried solvents or reagents. The catalyst is easily recovered and can be regenerated making the procedure economically viable even for large-scale synthesis.     

Differential processing of mammalian l-histidine decarboxylase enzymes

In the mammalian species studied so far, the l-histidine decarboxylase (HDC) enzyme responsible for histamine biosynthesis has been shown to undergo post-translational processing. The processing is best characterized for the mouse enzyme, where di-asparate DD motifs mediate the production of active ∼55 and ∼60 kDa isoforms from the ∼74 kDa precursor in a caspase-9 dependent manner. The identification of conserved di-aspartate motifs at similar locations in the rat and human HDC protein sequences has led to proposals that these may represent important processing sites in these species also. Here we used transfected Cos7 cells to demonstrate that the rat and human HDC proteins undergo differential processing compared to each other, and found no evidence to suggest that conserved di-aspartate motifs are required absolutely for processing in this cell type. Instead we identified SKD and EEAPD motifs that are important for caspase-6 dependent production of ∼54 and ∼59 kDa isoforms in the rat and human proteins, respectively. The addition of staurosporine, which is known to pharmacologically activate caspase enzymes, increased processing of the human HDC protein. We propose that caspase-dependent processing is a conserved feature of mammalian HDC enzymes, but that proteolysis may involve different enzymes and occur at diverse sites and sequences.     

Biofunctionalization of cellulosic fibres with l-cysteine: Assessment of antibacterial properties and mechanism of action against Staphylococcus aureus and Klebsiella pneumoniae

The main purpose of this work is to obtain a cotton-based textile material functionalized with l-cysteine (l-cys) to achieve an antimicrobial effect with potential application in biomedical, geriatric or pediatric textiles. The binding capacity of l-cys to cotton fibres was assessed through different functionalization strategies—surface activation and exhaustion processes. A subsequent analysis of the possible antibacterial action against Staphylococcus aureus and Klebsiella pneumoniae was performed according with the Japanese International standard ( JISL, 2008). To determine the mechanism of action of l-cys on the selected strains, flow cytometry was used.     

Distribution of radiolabeled l-glutamate and d-aspartate from blood into peripheral tissues in naive rats: Significance for brain neuroprotection

Excess l-glutamate (glutamate) levels in brain interstitial and cerebrospinal fluids (ISF and CSF, respectively) are the hallmark of several neurodegenerative conditions such as stroke, traumatic brain injury or amyotrophic lateral sclerosis. Its removal could prevent the glutamate excitotoxicity that causes long-lasting neurological deficits. As in previous studies, we have established the role of blood glutamate levels in brain neuroprotection, we have now investigated the contribution of the peripheral organs to the homeostasis of glutamate in blood. We have administered naive rats with intravenous injections of either l-[1-¹⁴C] Glutamic acid (l-[1-¹⁴C] Glu), l-[G-³H] Glutamic acid (l-[G-³H] Glu) or d-[2,3-³H] Aspartic acid (d-[2,3-³H] Asp), a non-metabolized analog of glutamate, and have followed their distribution into peripheral organs. We have observed that the decay of the radioactivity associated with l-[1-¹⁴C] Glu and l-[G-³H] Glu was faster than that associated with glutamate non-metabolized analog, d-[2,3-³H] Asp. l-[1-¹⁴C] Glu was subjected in blood to a rapid decarboxylation with the loss of ¹⁴CO₂. The three major sequestrating organs, serving as depots for the eliminated glutamate and/or its metabolites were skeletal muscle, liver and gut, contributing together 92% or 87% of total l-[U-¹⁴C] Glu or d-[2,3-³H] Asp radioactivity capture. l-[U-¹⁴C] Glu and d-[2,3-³H] Asp showed a different organ sequestration pattern. We conclude that glutamate is rapidly eliminated from the blood into peripheral tissues, mainly in non-metabolized form. The liver plays a central role in glutamate metabolism and serves as an origin for glutamate metabolites that redistribute into skeletal muscle and gut. The findings of this study suggest now that pharmacological manipulations that reduce the liver glutamate release rate or cause a boosting of the skeletal muscle glutamate pumping rate are likely to cause brain neuroprotection.     

Effects of N-acetyl-l-cysteine on adhesive strength between breast cancer cell and extracellular matrix proteins after ionizing radiation

Aims: To evaluate the effect of N-acetyl-L-cysteine (LNAC), a common ROS scavenger, on the adhesive affinity between MDA-MB-231 breast cancer cells and extracellular matrix (ECM) proteins after IR.     

Protein l-isoaspartyl-O-methyltransferase of Vibrio cholerae: Interaction with cofactors and effect of osmolytes on unfolding

Protein l-isoaspartyl-O-methyltransferase (PIMT) is an ubiquitous enzyme widely distributed in cells and plays a role in the repair of deamidated and isomerized proteins. In this study, we show that this enzyme is present in cytosolic extract of Vibrio cholerae, an enteric pathogenic Gram-negative bacterium and is enzymatically active. Additionally, we focus on the detailed biophysical characterization of the recombinant PIMT from V. cholerae to gain insight into its structure, stability and the cofactor binding. The equilibrium denaturation of PIMT has been studied using tryptophan fluorescence and CD spectroscopy. The far- and near-UV CD, as well as fluorescence experiments reveal the presence of a non-native intermediate in the folding pathway. Binding of the hydrophobic fluorescent probe, bis-ANS, to the intermediate occurs with high affinity because of the exposure of the hydrophobic clusters during the unfolding process. The existence of the probable intermediate has also been confirmed from limited tryptic digestion and DLS experiments. The protein shows higher binding affinity for AdoHcy, in comparison to AdoMet, and the binding increases the midpoint of thermal unfolding by 6 and 5 °C, respectively. Modeling and molecular dynamics simulations also support the higher stability of the protein in presence of AdoHcy.     

Influence of l-arginine supplementation on reproductive blood flow and embryo recovery rates in mares

Supplementation with l-arginine can increase uterine arterial blood flow and vascular perfusion of the preovulatory follicle in mares. Increased vascular perfusion of the preovulatory follicle has been correlated with successful pregnancy in mares. The objective of this study was to determine if supplemental l-arginine would increase ovarian arterial blood flow, vascular perfusion of the preovulatory follicle, and embryo recovery rates in mares. Mares were blocked by age and breed and assigned at random within block to l-arginine supplementation or control groups. Mares were fed l-arginine beginning 17 days before and through the duration of the study. Transrectal Doppler ultrasonography was used to measure ovarian arterial blood flow and vascular perfusion of the preovulatory follicle daily when it reached 35 mm and subsequent CL on Days 2, 4, and 6. Mares, on achieving a follicle of 35 mm or more were bred via artificial insemination and an embryo collection was attempted 7 days after ovulation. Treatment did not affect interovulatory interval (arginine-treated, 18.1 ± 2.6 days; control, 20.7 ± 2.3 days) or embryo recovery rate (arginine-treated, 54%; control, 48%). Mares treated with l-arginine had a larger follicle for the 10 days preceding ovulation than control mares (30.4 ± 1.2 and 26.3 ± 1.3 mm, respectively; P < 0.05) and vascular perfusion of the dominant follicle tended (P = 0.10) to be greater for the 4 days before ovulation. No differences were observed between groups in diameter or vascular perfusion of the CL. Resistance indices, normalized to ovulation, were not significantly different between groups during the follicular or luteal phase. Oral l-arginine supplementation increased the size and tended to increase perfusion of the follicle 1, but had no effect on luteal perfusion or embryo recovery rates in mares.     

Synthesis of xylan-graft-poly(l-lactide) copolymers via click chemistry and their thermal properties

Di-O-(6-azidohexanoyl)-xylan-graft-poly(l-lactide)s (XylC6N₃-g-PLLAs) were prepared by grafting propargyl-terminated poly(l-lactide) onto di-O-(6-azidohexanoyl)-xylan (XylC6N₃) via click chemistry. Di-O-(6-azidohexanoyl)-xylan (XylC6N₃) was prepared via two steps from xylan extracted from eucalyptus kraft pulp with aqueous sodium hydroxide solution. Propargyl-terminated poly(l-lactide)s (PLLA) with three different molecular weights were synthesized via ring-opening polymerization of l-lactide using propargyl alcohol as initiator and tin (II) octanoate (Sn(Oct)₂) as catalyst. XylC6N₃ and propargyl-terminated PLLAs were treated with N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) and copper(I) bromide, and the graft copolymers XylC6N₃-g-PLLAs were obtained. DSC measurements revealed that the glass transition temperatures (T_g) of the copolymers decreased compared to that of XylC6N₃, suggesting that the grafted PLLA side-chains act as an internal plasticizer for xylan. TGA measurements revealed that XylC6N₃-g-PLLAs had higher decomposition temperatures than those of XylC6N₃ or PLLA, and that the decomposition temperatures of the copolymers increased with decrease in the number of PLLA side-chains grafted to the xylan main-chain.     

Thermo-sensitivity and triggered drug release of polysaccharide nanogels derived from pullulan-g-poly(l-lactide) copolymers

Thermo-responsive nanogels from poly(l-lactide)-g-pullulan (PLP1 and 2) copolymers with different lactide contents were investigated as an anticancer drug delivery carrier. The phase transition temperature of PLP 1 with lower lactide content in distilled water showed around 35 °C. Upon adding 0.15 M NaCl to PLP 1, a significant difference in the transmittance was observed when comparing the non-additive salt condition. The total amount of released doxorubicin (DOX) from the DOX-loaded PLP nanogels increased with increasing temperature for 50 h. A noticeable difference in the initial release by PLP 1 was observed between 37 and 42 °C. In the 50% inhibitory concentration (IC₅₀) analysis, the IC₅₀ values of DOX released from PLP 1 were approximately 5.9 and 9.3 μg/mL at 37 and 42 °C, respectively. The results suggest that self-assembled PLP nanogels, by means of a triggering temperature, can be used as a long-term drug delivery system in cancer treatments.     

Separate binding sites in rat brain synaptic membranes for l-cysteine sulfinate and for l-glutamate

Binding of l-[³H]cysteine sulfinic acid (CSA) and l-[³H]glutamate were compared in various subcellular fractions and in the presence of a variety of pharmacological and ionic manipulations in order to test the possibility that the two amino acids possessed separate binding sites.The specific l-[³H]cysteine sulfinate binding was found to be enriched maximally in medium and high density synaptic membranes, while the crude mitochondrial synaptosomal fraction displayed the highest l-[³H]glutamate binding. The ratio of l-[³H]cysteine sulfinate binding/l-[³H]glutamate binding was variable across brain regions. Several compounds differentially affected l-[³H]cysteine sulfinate and l-[³H]glutamate binding. l-cysteine sulfinate was the most potent displacer regardless of the binding considered. Finally, while cations produced qualitatively similar effects on the binding of the two amino acids, quantitative differences were evident.In sum, these data revealed the complexity of l-[³H]cysteine sulfinate and l-[³H]glutamate binding. They suggest the existence of several binding sites and that some of these are shared by both substances. However, the results also indicate that separate binding sites for the two amino acids exist in synaptic membrane, giving further support to the hypothesis that cysteine sulfinate serves a neurotransmitter role in the central nervous system.     

Defining a kinetic mechanism for l-DOPA 2,3 dioxygenase,a single-domain type I extradiol dioxygenase from Streptomyces lincolnensis

l-DOPA-2,3-dioxygenase from Streptomyces lincolnensis is a single domain type I extradiol dioxygenase of the vicinal oxygen chelate superfamily and catalyzes the second step in the metabolism of the propylhygric acid moiety of the antibiotic, lincomycin. In this report, the kinetic mechanism of l-DOPA dioxygenase is interrogated using stopped-flow in order to determine microscopic rate constants. Pre-steady state, progress curve and steady-state data were combined in a global kinetic analysis using KinTek Explorer in order to define and constrain a kinetic model for the type I l-DOPA dioxygenase. The data are best described by a four step mechanism, in which the cyclization of the enzymatic product is not enzyme catalyzed.     

Cysteine desulfhydrase activity in cucurbit plants: Stimulation by preincubation with l- or d-cysteine

Cysteine desulfhydrase activity in leaf discs of cucurbit plants is enhanced 2–4-fold by preincubation with l or d-cysteine. Preincubation with structural analogs of cysteine also stimulated the activity of the enzyme, but to a smaller extent. Maximal increase in cysteine desulfhydrase activity was observed by preincubation with 5 mM or higher concentrations of cysteine. Although not caused by activation, stimulation of the enzyme activity was half-maximal within less than 15 min. Whereas the increase in cysteine desulfhydrase activity by preincubation of leaf discs with cysteine was light independent, pretreatment of the entire plant with light or dark determined the leaf discs' potential for stimulation of the enzyme. Exposure to darkness for 4 hr reduced this potential by 60%. It is concluded that the potential for stimulation of cysteine desulfhydrase activity by preincubation with cysteine is regulated by a compound not synthesized, but metabolized, in the leaf tissue. This regulatory compound may be supplied to the leaves by long-distance transport.     

Pyrrolizidine alkaloid biosynthesis: Derivation of retronecine from l-arginine and l-ornithine

The relative retention of ³H and ¹⁴C on incorporation of d-, l- and dl-isomers of [¹⁴C]arginine and [¹⁴C]ornithine into retrorsine using L-[5-³H]arginine as an internal standard has been measured. The retronecine portion of the pyrrolizidine alkaloid retrorsine, present in Senecio isatideus plants, is shown to be derived from l-arginine and l-ornithine.     

Alkyl alkanethiolsulfonate sulfhydryl reagents: β-Sulfhydryl-modified derivatives of l-cysteine as substrates for trypsin and α-chymotrypsin

Derivatives of l-cysteine and the A chain of bovine insulin have been chemically modified at the cysteinyl β-sulfhydryl by certain sulfhydryl-specific alkyl alkanethiolsulfonate reagents. The alkanethiolation products possess mixed-disulfide side chains structurally similar to the side chains of lysine and phenylalanine and hence were studied here as substrates for trypsin and α-chymotrypsin, respectively. Kinetic parameters were obtained for the enzyme-catalyzed hydrolyses of the modified l-cysteine analogs and of specific reference amino acids which were derivatized analogously at both the α-amino and α-carboxyl groups and assayed identically. For both enzymes it was found that the specificity constants, $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, for analog esters compare favorably with those for specific reference esters, whereas specificity constants for analog amides compare much less favorably with those for specific reference amides. This discrepancy is largely a consequence of the k_cat values for the analog amides being relatively much lower than the corresponding values for the reference amides. Consistent with this trend, no detectable enzyme-catalyzed hydrolysis of the amide bonds at the sites of modified cysteine residues in the A chain of bovine insulin was observed. It is proposed that the predominant kinetic consequence of the mixed-disulfide side chains of the alkanethiolated cysteine moieties is a decrease in the acylation rate constants, k₂, arising from an increase in the transition-state free energies of acylation.     

Prevention of DNA damage by l-carnitine induced by metabolites accumulated in maple syrup urine disease in human peripheral leukocytes in vitro

Maple syrup urine disease (MSUD) is an inherited aminoacidopathy caused by a deficiency in branched-chain α-keto acid dehydrogenase complex activity that leads to the accumulation of the branched-chain amino acids (BCAAs) leucine (Leu), isoleucine, and valine and their respective α-keto-acids, α-ketoisocaproic acid (KIC), α keto-β-methylvaleric acid, and α-ketoisovaleric acid. The major clinical features presented by MSUD patients include ketoacidosis, failure to thrive, poor feeding, apnea, ataxia, seizures, coma, psychomotor delay, and mental retardation; however, the pathophysiology of this disease is poorly understood. MSUD treatment consists of a low protein diet supplemented with a mixture containing micronutrients and essential amino acids but excluding BCAAs. Studies have shown that oxidative stress may be involved in the neuropathology of MSUD, with the existence of lipid and protein oxidative damage in affected patients. In recent years, studies have demonstrated the antioxidant role of l-carnitine (l-Car), which plays a central function in cellular energy metabolism and for which MSUD patients have a deficiency. In this work, we investigated the in vitro effect of Leu and KIC in the presence or absence of l-Car on DNA damage in peripheral whole blood leukocytes using the alkaline comet assay with silver staining and visual scoring. Leu and KIC resulted in a DNA damage index that was significantly higher than that of the control group, and l-Car was able to significantly prevent this damage, mainly that due to KIC.     

Sister-chromatid exchange induced by X-ray of human lymphocytes and the effect of l-cysteine

A staining technique that detects sister-chromatid exchanges (SCEs) has been used to examine the response of human lymphocyte chromosomes to various dosages of X-irradiation. The SCE frequency was markedly increased following irradiation. However, the increase was of a significantly smaller magnitude when irradiation occurred in the presence of an antimutagenic agent. Scoring SCEs may provide a useful technique for assaying the mutagenic effects of environmental carcinogens as well as the protective effects of antimutagenic agents.     

Measurements of serum glucose using the luciferin/luciferase system and a liquid scintillation spectrometer

A single-step assay for serum glucose measurements is described. The assay is based on the phosphorylation of D-glucose by glucokinase and the measurement of ATP consumption by firefly luciferase. The luminescence is recorded in an ordinary liquid scintillation spectrometer. The use of stable reagents and a stable final signal (light emission) makes it possible to analyze a large number of samples in each assay run. The assay is of particular value when repeated serum glucose determinations are performed on samples from small laboratory animals.     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium