Applications of luciferin biosynthesis: Bioluminescence assays for l-cysteine and luciferase

Based on the biosynthetic pathway of firefly bioluminescence substrate d-luciferin, the concentration of l-cysteine can be quantified using a simple protocol and a conventional luminescence detector. The lower limit of quantification (signal/noise ratio [S/N] = 10) was 0.26 μM. Using our method, the total amount of free/reduced and disulfide/oxidized l-cysteine could be measured successfully in human serum. In addition, biosynthetic precursors such as 2-cyano-6-hydroxybenzothiazole and l-luciferin could replace d-luciferin in the cell-based luciferase assay. Our results suggest that the bioluminescence reaction associated with the biosynthesis of bioluminescence substrates can provide a fast and cost-effective assay method.     

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.     

Estrogen regulates the expression of N-methyl-d-aspartate (NMDA) receptor subunit epsilon 4 (Grin2d), that is essential for the normal sexual behavior in female mice

Estrogen plays important roles in the reproductive behavior of animals. In the present study, we found that the Grin2d gene of mouse possessed half-sites of the estrogen-responsive element (ERE) in the 3′-untranslated region (UTR). Quantitative PCR analysis showed that the reduced Grin2d mRNA expression in the hypothalamus of the ovariectomized mice was restored by estrogen administration. Downregulation of Grin2d mRNA expression was also detected in the hypothalamus of estrogen receptor alpha-knockout female mice. Moreover, estrogen-induced lordosis response was decreased in Grin2d-knockout mice. These results suggest that estrogen regulates lordosis behavior through the regulation of Grin2d expression in the hypothalamus of female mice.     

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.     

Ginsenoside Rg1 provides neuroprotection against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion through downregulation of aquaporin 4 expression

Ginsenoside Rg1 is regarded as one of main bioactive compounds responsible for pharmaceutical actions of ginseng with little toxicity and has been shown to have possibly neuroprotective effects. However, the mechanism of its neuroprotection for acute ischemic stroke is still elusive. The purpose of present study is thus to assess the neuroprotective effects of the ginsenoside Rg1 against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion, and then to explore the mechanisms for these neuroprotective effects by targeting aquaporin 4. Focal cerebral ischemia was induced by middle cerebral artery occlusion. Neurological examinations were performed by using Longa's 5-point scale. Evans blue dye was used to investigate the effects of ginsenoside Rg1 on blood brain barrier permeability. Immunohistochemical analysis and real-time fluorescence quantitative polymerase chain reaction were used to assess aquaporin 4 expression. As a result, general linear model with repeated measures analysis of variance for neurological scores at 5 repeated measures showed that ginsenoside Rg1-treated group could significantly reduce the changing trend of neurological deficit scores when compared with the middle cerebral artery occlusion model group (p < 0.05). Compared with the middle cerebral artery occlusion model group, ginsenoside Rg1 group has significantly decreased Evans blue content and reduced aquaporin 4 expression at each time point (p < 0.05). In conclusion, ginsenoside Rg1 as a ginsenoside neuroprotective agent could improve neurological injury, attenuate blood brain barrier disruption and downregulate aquaporin 4 expression induced by cerebral ischemia/reperfusion insults in rats.     

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.     

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.     

The crystal structure of d-mandelate dehydrogenase reveals its distinct substrate and coenzyme recognition mechanisms from those of 2-ketopantoate reductase

d-Mandelate dehydrogenases (d-ManDHs), belonging to a new d-2-hydroxyacid dehydrogenase family, catalyze the conversion between benzoylformate and d-mandelate using NAD as a coenzyme. We determined the first d-ManDH structure, that of ManDH2 from Enterococcus faecalis IAM10071. The overall structure showed ManDH2 has a similar fold to 2-ketopantoate reductase (KPR), which catalyzes the conversion of 2-ketopantoate to d-pantoate using NADP as a coenzyme. They share conserved catalytic residues, indicating ManDH2 has the same reaction mechanism as KPR. However, ManDH2 exhibits significant structural variations in the coenzyme and substrate binding sites compared to KPR. These structural observations could explain their different coenzyme and substrate specificities.     

Bioanalysis of 6-diazo-5-oxo-l-norleucine in plasma and brain by ultra-performance liquid chromatography mass spectrometry

Glutamine is an abundant amino acid that plays pivotal roles in cell growth, cell metabolism, and neurotransmission. Dysregulation of glutamine-using pathways has been associated with pathological conditions such as cancer and neurodegenerative diseases. 6-Diazo-5-oxo-l-norleucine (DON) is a reactive glutamine analog that inhibits enzymes affecting glutamine metabolism such as glutaminase, 2-N-amidotransferase, l-asparaginase, and several enzymes involved in pyrimidine and purine de novo synthesis. As a result, DON is actively used in preclinical models of cancer and neurodegenerative disease. Moreover, there have been several clinical trials using DON to treat a variety of cancers. Considerations of dose and exposure are especially important with DON treatment due to its narrow therapeutic window and significant side effects. Consequently, a robust quantification bioassay is of interest. DON is a polar unstable molecule that has made quantification challenging. Here we report on the characterization of a bioanalytical method to quantify DON in tissue samples involving DON derivatization with 3 N HCl in butanol. The derivatized product is lipophilic and stable. Detection of this analyte by mass spectrometry is fast and specific and can be used to quantify DON in plasma and brain tissue with a limit of detection at the low nanomolar level.     

外科血培养标本中病原菌分布及耐药性分析

目的了解中山大学附属第一医院外科血标本中病原菌的菌种分布及常见菌株的耐药性。方法血标本用Bact/A lert-120全自动血培养仪进行血培养,阳性血培养转种后用VITEK-60 AMS细菌鉴定仪鉴定,用K-B法进行药敏试验。结果2002年1月至2005年12月血培养标本中共分离出病原菌256株,阳性率为10.6%。122株(47.7%)为革兰阴性杆菌,其中肠杆菌科细菌占72.1%(88/122),非发酵菌占27.9%(34/122);113株(44.1%)为革兰阳性球菌,其中葡萄球菌属占51.3%(58/113),肠球菌占38.1%(43/113);真菌21株(8.2%)。血培养中的革兰阴性杆菌对亚胺培南、美洛培南治疗敏感;革兰阳性球菌对万古霉素和替考拉宁敏感。结论肠杆菌科细菌和葡萄球菌是外科血培养中的主要病原菌,其耐药现象严重,宜根据药敏结果选用敏感抗菌药物治疗。     

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.     

Quantitative autoradiographic characterization of receptors for angiotensin II and other neuropeptides in individual brain nuclei and peripheral tissues from single rats

Autoradiographic techniques coupled with computerized microdensitometry and comparison with 125I standards were used to characterize and quantitate receptors for neuropeptides in rat brain and adrenal and pituitary glands. These techniques are rapidly performed, anatomically precise, and more sensitive than membrane binding techniques. They permit the determination of complete saturation curves and Scatchard analysis in discrete nuclei of the rat brain and in single rat pituitary and adrenal glands. Angiotensin II (AII) receptors were quantitated after incubation of 16-micron tissue sections with the AII agonist 125I-[Sar1]-AII. High-affinity, high-density AII receptors were present in the organon subfornicalis, organon vasculosum laminae terminalis and nuclei triangularis septalis, suprachiasmatis, and paraventricularis of the rat and in rat adrenal capsule-zona glomerulosa area, adrenal medulla, and anterior pituitary. These techniques could be used for precise localization and quantitation of other neuropeptide receptors in single rat brain nuclei, after optimizing the assay conditions and provided that suitable 125I ligands are available.     

Incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into proteins and lipid intermediates in microsomal and golgi membranes from rat liver

Rough and smooth microsomes and Golgi membranes incorporate $\text{N-}\text{acetylglucosamine}$ from $\text{UDP}\text{-N-}\text{acetylglucosamine}$ into endogenous protein acceptors. A lipid intermediate of the dolichol phosphate type participates in this transfer reaction in the case of both microsomal subfractions, but the nature of lipid glycosylation is different in these two fractions. Glucosamine transfer in Golgi membranes does not appear to involve a lipid intermediate. In contrast to the results obtained under in vivo conditions, no glucosamine label is recovered in nascent ribosomal proteins or on luminal secretory proteins after incubation in vitro. Proteolysis of intact vesicles of the subfractions removes glycosylated dolichol phosphate and protein acceptors to various extents and interferes with transferase activities. This finding suggests the possibility that glycosylation at the cytoplasmic side of the membrane of the endoplasmic reticulum may involve a system separate from that acting at the luminal side of the same membrane.     

Redox atlas of the mouse : Immunohistochemical detection of glutaredoxin-, peroxiredoxin-, and thioredoxin-family proteins in various tissues of the laboratory mouse

Background

Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available.

Methods

Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry.

Results

The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues.

Conclusion

Our results imply more specific functions and interactions between the proteins of this family than previously assumed.

General significance

Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases—the laboratory mouse.This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.     

Comparison of PCR and culture for detection of Nocardia asteroides in brain specimens from experimentally infected BALB/c mice

Systemic infection of BALB/c mice with Nocardia asteroides strain GUH-2 results in widespread replication of the organism in the brain, followed by its immune-mediated clearance. The present study compared the sensitivity of polymerase chain reaction (PCR) to bacterial culture for detection of cerebral nocardial infection in this experimental system. Mice (n=4/time point) were administered N. asteroides by intravenous injection, and brain specimens were evaluated for Nocardia by PCR and culture at post-infection days 2, 7, 14 and 21. Nocardia was detected by PCR in all infected animals on post-infection days 2, 7, and 14, and in one of four mice on post-infection day 21; in contrast, the organism was detected by culture only on post-infection days 2 and 7. These findings suggest that PCR may be more sensitive than culture for the detection of low numbers of Nocardia in the brain.     

Secondary structure of peptides: 8. 13C n.m.r. CP/MAS investigation of solid poly(l-leucines) and poly(d-norvalines) prepared from N-carboxyanhydrides

¹³C n.m.r. CP/MAS spectra (50.3 and 75.4 MHz) of solid poly(l-lleucines) and poly(d-norvalines) measured with suitable acquisition parameters allow quantification of the composition of the secondary structure. The optimum acquisition parameters were found by systematic variation of the contact time by means of samples containing 5?0% α-helix structure. The polypeptides were prepared by primary or tertiary amine-initiated polymerizations of the corresponding amino acid NCAs and the average degrees of polymerization ($\text{DP}$) were determined by ¹H n.m.r. endgroup analysis. The mole fraction of α-helices increases with increasing $\text{DP}$; it depends on the nature of the solvent and to a lesser degree on the polymerization temperature. When prepared under identical conditions, poly(d-norvaline) samples contain more β-sheet structure than poly(l-leucine. Reprecipitation increases the α-helix content, demonstrating that a part of the original β-sheet structure is thermodynamically unstable. The presence of oligomers of $\text{DP}$ ?10 is mainly responsible for the thermodynamically stable part of the β-sheet structure. The chain growth mechanism is discussed.     

Significance of magnetic resonance image and blood manganese measurement for the assessment of brain manganese during total parenteral nutrition in rats

In this study, we report on the influence of trace elements (TE) on signal intensities of nuclear magnetic resonance images (MRI), both in vivo and in vitro. Optimal parameters for the assessment of Mn concentration in the brain of rats on total parenteral nutrition were established. For the in vitro study, Mn and trace element solutions, one containing Zn, Cu, Fe, and I (TE-4) and another containing the above elements plus Mn (TE-5), were diluted with physiological saline or with rat brain homogenate and used to measure signal intensities in MRI. Concentration-dependent signal hyperintensity was observed in both cases in the Mn and the TE-5 solutions, but no effect was observed with the TE-4 solution. The signal increase was greater for brain tissue homogenates. In the in vivo study, the experimental animals were maintained under total parenteral nutrition (TPN) with a standard clinical dose of TE-5 and/or with 10-fold the clinical dose of TE-4 and TE-5 for 1 wk. Only rats that were receiving the increased TE-5 dose showed signal hyperintensity on MRI. Positive correlations were observed among the signal hyperintensity, the blood Mn concentrations, and that of the rat brain. Our results suggest that Mn in TE preparations may be the cause of signal hyperintensity on MRI in a concentration-dependent fashion, and that MRI and measurement of blood Mn may be used to estimate Mn accumulation in brain tissue.     

Regulation of the cancer cell membrane lipid composition by NaCHOleate : Effects on cell signaling and therapeutical relevance in glioma

This review summarizes the cellular bases of the effects of NaCHOleate (2-hydroxyoleic acid; 2OHOA; Minerval) against glioma and other types of tumors. NaCHOleate, activates sphingomyelin synthase (SGMS) increasing the levels of cell membrane sphingomyelin (SM) and diacylglycerol (DAG) together with reductions of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). The increases in the membrane levels of NaCHOleate itself and of DAG induce a translocation and overexpression of protein kinase C (PKC) and subsequent reductions of Cyclin D, cyclin-dependent kinases 4 and 6 (CDKs 4 and 6), hypophosphorylation of the retinoblastoma protein, inhibition of E2F1 and knockdown of dihydrofolate reductase (DHFR) impairing DNA synthesis. In addition in some cancer cells, the increases in SM are associated with Fas receptor (FasR) capping and ligand-free induction of apoptosis. In glioma cell lines, the increases in SM are associated with the inhibition of the Ras/MAPK and PI3K/Akt pathways, in association with p27Kip1 overexpression. Finally, an analysis of the Repository of Molecular Brain Neoplasia Data (REMBRANDT) database for glioma patient survival shows that the weight of SM-related metabolism gene expression in glioma patients' survival is similar to glioma-related genes. Due to its low toxicity and anti-tumoral effect in cell and animal models its status as an orphan drug for glioma treatment by the European Medicines Agency (EMA) was recently acknowledged and a phase 1/2A open label, non-randomized study was started in patients with advanced solid tumors including malignant glioma. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.