K+-dependent 3H-d-glucose transport by hepatopancreatic brush border membrane vesicles of a marine shrimp

The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-d-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl₂/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-d-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-d-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J _max and lowered K_m in both salts. ³H-d-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis–Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-d-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (d-galactose, α-methyl-d-gluco-pyranoside, unlabeled d-glucose, d-fructose, and d-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-d-glucose influx. Only unlabeled d-glucose, d-fructose, and d-mannose significantly (p < 0.05) reduced labeled glucose transport. An additional experiment using increasing concentrations of d-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-d-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-d-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, d-fructose, and d-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter, suggesting that an invertebrate analogue of this protein may occur in shrimp.     

Backbone and side-chain 1H, 15N and 13C assignment of apo- and imipenem-acylated l,d-transpeptidase from Bacillus subtilis

The d,d-transpeptidase activity of Penicillin Binding Proteins (PBPs) is essential to maintain cell wall integrity. PBPs catalyze the final step of the peptidoglycan synthesis by forming 4 → 3 cross-links between two peptide stems. Recently, a novel β-lactam resistance mechanism involving l,d-transpeptidases has been identified in Enterococcus faecium and Mycobacterium tuberculosis. In this resistance pathway, the classical 4 → 3 cross-links are replaced by 3 → 3 cross-links, whose formation are catalyzed by the l,d-transpeptidases. To date, only one class of the entire β-lactam family, the carbapenems, is able to inhibit the l,d-transpeptidase activity. Nevertheless, the specificity of this inactivation is still not understood. Hence, the study of this new transpeptidase family is of considerable interest in order to understand the mechanism of the l,d-transpeptidases inhibition by carbapenems. In this context, we present herein the backbone and side-chain ¹H, ¹⁵N and ¹³C NMR assignment of the l,d-transpeptidase from Bacillus subtilis (Ldt_Bs) in the apo and in the acylated form with a carbapenem, the imipenem.     

Orientation of the tryptophanyl side chain in [(d)TrpA1] and [TrpA1]insulin

Insulin analogues withl- andd-tryptophan instead of glycine in A1 permit an estimate of the proximity relationship between the indole residue of tryptophan and B19-tyrosine by evaluation of singlet-singlet resonance energy transfer. A significantly higher transfer efficiency is observed with [(d)Trp^A1]insulin than with the [Trp^A1]analogue. On the basis of this result it is possible to deduce the arrangement of the side chains and the α-amino groups in position A1 of [(d)Trp^A1] and [Trp^A1]insulin.     

Cellular Senescence Induced by Prolonged Subculture Adversely Affects Glutamate Uptake in C6 Lineage

Several researchers have recently used C6 cells to evaluate functional properties of high-affinity glutamate transporters. However, it has been demonstrated that this lineage suffers several morphological and biochemical alterations according to the number of passages in culture. Currently, there are no reports showing whether functional properties of high-affinity glutamate transporters comply with these sub culturing-dependent modifications. The present study aimed to compare the functional properties of high-affinity glutamate transporters expressed in early (EPC6) and late (LPC6) passage C6 cells through a detailed pharmacological and biochemical characterization. Between 60–180 min of l-[³H]glu incubation, LPC6 presented an intracellular [³H] 55 % lower than EPC6. Both cultures showed a time-dependent increase of intracellular [³H] reaching maximal levels at 120 min. Cultures incubated with d-[³H]asp showed a time-dependent increase of [³H] until 180 min. Moreover, LPC6 have a d-[³H]asp-derived intracellular [³H] 30–45 % lower than EPC6 until 120 min. Only EAAT3 was immunodetected in cultures and its total content was equal between them. PMA-stimulated EAAT3 trafficking to membrane increased 50 % of l-[³H]glu-derived intracellular [³H] in EPC6 and had no effect in LPC6. LPC6 displayed characteristics that resemble senescence, such as high β-Gal staining, cell enlargement and increase of large and regular nuclei. Our results demonstrated that LPC6 exhibited glutamate uptake impairment, which may have occurred due to its inability to mobilize EAAT3 to cell membrane. This profile might be related to senescent process observed in this culture. Our results suggest that LPC6 cells are an inappropriate glial cellular model to investigate the functional properties of high-affinity glutamate transporters.     

Transportable and Non-transportable Inhibitors of L-glutamate Uptake Produce Astrocytic Stellation and Increase EAAT2 Cell Surface Expression

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (d-Aspartate (d-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (l-CCG-III)) and non-transportable (dl-threo-β-benzyloxyaspartate (dl-TBOA)) inhibitors of Glu uptake in murine astrocytes. d-Asp (1 mM), l-CCG-III (0.5 mM) and dl-TBOA (0.5 mM) produced time-dependent (24–72 h) reductions in ³[H]d-Asp uptake (approximately 30–70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for l-CCG-III and dl-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.     

Determination of two sites of automethylation in bovine erythrocyte protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase

Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed theαPCM fraction [Lindquist and McFadden (1994),J. Protein Chem. 13, 23–30]. The altered aspartyl sites serving as methyl acceptors inαPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [³H]automethylated enzyme that contain a [³H]methyl ester. Methylation was positively identified at positions Asn₁₈₈ and Asp₂₁₇ in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatαPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

An Acute Glutamate Exposure Induces Long-Term Down Regulation of GLAST/EAAT1 Uptake Activity in Cultured Bergmann Glia Cells

Glutamate, the major excitatory neurotransmitter in the vertebrate brain, is a potent neurotoxin therefore its extracellular levels have to be tightly regulated by means of sodium-dependent glutamate uptake systems of the slc1A family. The glial glutamate/aspartate transporter (GLAST/EAAT1) and the glutamate transporter 1 carry most of the uptake activity in cerebellum and in the forebrain, respectively. In the cerebellar cortex, GLAST is profusely expressed in Bergmann glia cells, which completely enwrap the parallel fiber-Purkinje cells synapses. Glutamate exposure in these cells, down regulates the activity as well as the expression levels of this transporter. In order to characterize the persistence of a single glutamate exposure, we followed the [³H]-d-aspartate uptake activity as a function of time after the removal of the glutamatergic stimulus. We were able to demonstrate that a single 30 min exposure to glutamate reduces the uptake activity for up to 3 h. This effect is dose-dependent and it is not reproduced neither by ionotropic nor metabotropic glutamate receptors agonists. In contrast, transporter specific ligands such as d-aspartate or l-(?)-threo-3-Hydroxyaspartic acid fully reproduce the glutamate effect. Equilibrium binding experiments revealed a decrease in [³H]-d-aspartate Bmax without a significant change in affinity, clearly suggesting that a reduction in the availability of plasma membrane glutamate transporters is the molecular basis of this effect. Interestingly, neither Glast mRNA nor its protein levels were significantly reduced upon the single glutamate exposure. Taken together, these results favor the notion of a transporter-mediated tight control of the uptake process.     

High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases

The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form d(?)-tartaric acid or l(+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[d] and CESH[l], producing d(?)- and l(+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver?CBurk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[l] showed much higher catalytic efficiency than CESH[d], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[l] and CESH[d] have different structural folds and potential active site residues. CESH[l] adopted a typical ??/??-hydrolase fold with a cap domain and a core domain, whereas CESH[d] possessed a unique TIM barrel fold composed of 8 ??-helices and 8 ??-strands, and 2 extra short ??-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[d], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs.     

Metabolic engineering of Escherichia coli for biosynthesis of d-galactonate

d-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert d-galactose into d-galactonate, a valuable compound in the polymer and cosmetic industries. d-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L^?1 d-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L^?1 d-galactonate. Inherent metabolic pathways for assimilating both d-galactose and d-galactonate were blocked to enhance the production of d-galactonate. This approach finally led to a 7.3-fold increase with d-galactonate concentration of 1.24 g L^?1 and yield of 62.0 %. Batch fermentation in 20 g L^?1 d-galactose of E. coli ?galK?dgoK mutant expressing the gld resulted in 17.6 g L^?1 of d-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of d-galactonate.     

Alteration of intrinsic amounts of d-serine in the mice lacking serine racemase and d-amino acid oxidase

For elucidation of the regulation mechanisms of intrinsic amounts of d-serine (d-Ser) which modulates the neuro-transmission of N-methyl-d-aspartate receptors in the brain, mutant animals lacking serine racemase (SRR) and d-amino acid oxidase (DAO) were established, and the amounts of d-Ser in the tissues and physiological fluids were determined. d-Ser amounts in the frontal brain areas were drastically decreased followed by reduced SRR activity. On the other hand, a moderate but significant decrease in d-Ser amounts was observed in the cerebellum and spinal cord of SRR knock-out (SRR^?/?) mice compared with those of control mice, although the amounts of d-Ser in these tissues were low. The amounts of d-Ser in the brain and serum were not altered with aging. To clarify the uptake of exogenous d-Ser into the brain tissues, we have determined the d-Ser of SRR^?/? mice after oral administration of d-Ser for the first time, and a drastic increase in d-Ser amounts in all the tested tissues was observed. Because both DAO and SRR are present in some brain areas, we have established the double mutant mice lacking SRR and DAO for the first time, and the contribution of both enzymes to the intrinsic d-Ser amounts was investigated. In the frontal brain, most of the intrinsic d-Ser was biosynthesized by SRR. On the other hand, half of the d-Ser present in the hindbrain was derived from the biosynthesis by SRR. These results indicate that the regulation of intrinsic d-Ser amounts is different depending on the tissues and provide useful information for the development of treatments for neuronal diseases.     

Cyclic dipeptides from rhabditid entomopathogenic nematode-associated Bacillus cereus have antimicrobial activities

The cell free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain four bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (FABMS, ¹H NMR, ¹³C NMR, ¹H–¹H COSY, ¹H–¹³C HMBC) and Marfey’s method. The compounds were identified as cyclic dipeptides (CDPs): cyclo(l-Pro-l-Trp), cyclo(l-Leu-l-Val), cyclo(d-Pro-d-Met), and cyclo(d-Pro-d-Phe), respectively. Compounds recorded significant antibacterial activity against all the test bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and methicillin-resistant S. aureus) except cyclo(l-Leu-l-Val). Cyclo(l-Leu-l-Val) recorded activity only against Gram positive bacteria. Best antibacterial activity was recorded by cyclo(l-Pro-l-Trp) against S. aureus (4 μg/ml). The four compounds were active against all the five fungi tested (Trichophyton rubrum, Aspergillus flavus, Candida albicans, Candida tropicalis and Cryptococcus neoformans) and the activity was compared with amphotericin B, the standard fungicide. The highest activity of 1 μg/ml by cyclo(l-Pro-l-Trp) was recorded against T. rubrum, a human pathogen responsible for causing athlete’s foot, jock itch, and ringworm. The activity of cyclo(l-Pro-l-Trp) against T. rubrum, C. neoformans and C. albicans were better than amphotericin B, the standard antifungal agent. To our knowledge, this is the first report of antifungal activity of CDPs against the human pathogenic fungi T. rubrum and C. neoformans. The four CDPs are nontoxic to healthy human cell line up to 200 μg/ml. We conclude that the bacterium associated with entomopathogenic nematode is promising sources of natural antimicrobial secondary metabolites, which may receive greater benefit as potential sources of new drugs in the pharmaceutical industry.     

Overexpression of d-psicose 3-epimerase from Ruminococcus sp. in Escherichia coli and its potential application in d-psicose production

The d-psicose 3-epimerase (DPE) gene from Ruminococcus sp. was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and characterized. It was optimally active at pH 7.5–8.0 and 60?°C. Activity was not dependent on the presence of metal ions; however, it became more thermostable with added Mn²⁺. The K _m of the enzyme for d-psicose (48?mM) was lower than that for d-tagatose (230?mM), suggesting that d-psicose is the optimum substrate. More importantly, the thermostability of the novel DPE from Ruminococcus is the strongest among all of the d-psicose and d-tagatose 3-epimerases and may be suitable for the industrial production of d-psicose from fructose.     

A d-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for d-psicose production: cloning, expression, purification, and characterization

d-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for d-psicose production from d-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a d-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co²⁺. Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using d-psicose as the substrate, the apparent K _m, k _cat, and catalytic efficiency (k _cat/K _m) were 27.4 mM, 49 s^?1, and 1.78 s^?1 mM^?1, respectively. Under the optimal conditions, the equilibrium ratio of d-fructose to d-psicose was 69:31. For high production of d-psicose, 216 g/L d-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate d-fructose, indicating that the enzyme was a potential d-psicose producer for industrial production.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

Synthesis of enantiomeric pureCis-dichloroplatinum(II) amino acid complexes

The reaction of potassium tetrachloroplatinate(II) with six representative sulfurcontaining amino acids, namely,d- andl-cysteine,d- andl-methionine and its methyl ester hydrochloride gives the corresponding enantiomerically purecis-dichloroplatinum(II) complexes. This represents the first reported series of well-characterized enantiomerically pure platinum(II) complexes for bothd- andl-amino acids. The spectroscopic properties, including IR,¹H-NMR, and¹³C NMR, of these complexes and their configuration are discussed.     

Amperometric microbiosensor as an alternative tool for investigation of d-serine in brain

This paper discusses the application of a reagentless, selective microbiosensor as a useful alternative tool for monitoring d-serine in neural samples. The main components of the 125-μm-diameter disk biosensor were d-amino acid oxidase for d-serine sensitivity (linear region slope, 61?±?7?μA?cm^–2?mM^–1; limit of detection, 20?nM), and poly-phenylenediamine for rejection of electroactive interference. The response time of the biosensor was of the order of 1?s, ideal for ‘real-time’ monitoring, and detection of systemically administered d-serine in brain extracellular fluid is demonstrated. Exploitation of this probe might resolve queries involving regulation of d-serine in excitotoxicity, and modulation of N-methyl-d-aspartate receptor function by d-serine and glycine in the central nervous system.     

The activity of erythrocyte and brain Na+/K+ and Mg2+-ATPases in rats subjected to acute homocysteine and homocysteine thiolactone administration

Hyperhomocysteinemia is associated with various pathologies including cardiovascular disease, stroke, and cognitive dysfunctions. Systemic administration of homocysteine can trigger seizures in animals, and patients with homocystinuria suffer from epileptic seizures. Available data suggest that homocysteine can be harmful to human cells because of its metabolic conversion to homocysteine thiolactone, a reactive thioester. A number of reports have demonstrated a reduction of Na⁺/K⁺-ATPase activity in cerebral ischemia, epilepsy and neurodegeneration possibly associated with excitotoxic mechanisms. The aim of this study was to examine the in vivo effects of d,l-homocysteine and d,l-homocysteine thiolactone on Na⁺/K⁺- and Mg²⁺-ATPase activities in erythrocyte (RBC), brain cortex, hippocampus, and brain stem of adult male rats. Our results demonstrate a moderate inhibition of rat hippocampal Na⁺/K⁺-ATPase activity by d,l-homocysteine, which however expressed no effect on the activity of this enzyme in the cortex and brain stem. In contrast,d,l-homocysteine thiolactone strongly inhibited Na⁺/K⁺-ATPase activity in cortex, hippocampus and brain stem of rats. RBC Na⁺/K⁺-ATPase and Mg²⁺-ATPase activities were not affected by d,l-homocysteine, while d,l-homocysteine thiolactone inhibited only Na⁺/K⁺-ATPase activity. This study results show that homocysteine thiolactone significantly inhibits Na⁺/K⁺-ATPase activity in the cortex, hippocampus, and brain stem, which may contribute at least in part to the understanding of excitotoxic and convulsive properties of this substance.