Isolation, identification and characterization of a novel antimicrobial peptide from Moringa oleifera seeds based on affinity adsorption.

This study aimed to characterize a novel antimicrobial peptide (AMP) obtained from Moringa oleifera seed protein hydrolysates. Cell membrane chromatography and live bacteria adsorption were combined into a single step to efficiently isolate the active fraction of the AMP. Five peptides were identified by LC-MS/MS, among which the MCNDCGA peptide (termed MOp3) showed the greatest inhibitory effect against Staphylococcus aureus [minimum inhibitory concentration (MIC): 2 mg/mL]. MOp3 was identified as a hydrophobic anionic AMP rich in ß-sheet structures with negligible hemolytic activity at 2.0 × MIC. MOp3 had good tolerance to salt solutions at 5 % and pH range 6.0-8.0, but was sensitive to high temperatures (>100 °C) and acid protease. Microscopic observation further revealed that MOp3 induced irreversible damage onto the cell membrane of S. aureus and interacted with dihydrofolate reductase and DNA gyrase by hydrogen bonding and hydrophobic interaction. These findings highlight the potential application of a new antimicrobial agent against S. aureus in the food industry.

Hydrophilic molecularly imprinted polymers functionalized magnetic carbon nanotubes for selective extraction of cyclic adenosine monophosphate from winter jujube.

In this work, a green strategy was developed to prepare molecularly imprinted polymers functionalized magnetic carbon nanotubes in aqueous phase under mild conditions for cyclic adenosine monophosphate. Thanks to water solubility of chitosan, a natural polysaccharide which is rich in amino and hydroxyl groups, provided the feasibility to synthesize the green molecularly imprinted polymers for water soluble template in aqueous media. Coupled with high-performance liquid chromatography, the method exhibited a short equilibrium time (6 min), high adsorption capacity (22.42 µg/mg), high magnetic susceptibility, and good selectivity to template molecule with the imprinting factor of 2.94. A good linearity in the range of 0.020-3.0 mg/mL for target was obtained with a correlation coefficient of 0.9998. The limit of detection (signal-to-noise ratio = 3) and limit of quantitation (signal-to-noise ratio = 10) of the magnetic solid phase extraction method for cyclic adenosine monophosphate were 5 and 15 ng/mg, respectively. And the practical application of chitosan-based molecularly imprinted polymers as adsorbent to isolate and determine cyclic adenosine monophosphate in real natural samples (winter jujube) was demonstrated.

Confined gold nanoparticles enhance the detection of small molecules in label-free impedance aptasensors.

A controlled architecture of nanoelectrodes, of a similar size to small molecule-binding aptamers, is synthesized inside nanoporous alumina. Gold nanoparticles with a controlled size (about 2 nm) are electrogenerated in the alumina cavities, showing a fast electron transfer process toward ferrocyanide. These uncapped nanoparticles are easily modified with a thiol-containing aptamer for label-free detection of adenosine monophosphate by electrochemical impedance spectroscopy. Our results show that the use of a limited electrical conducting surface inside an insulating environment can be very sensitive to conformational changes, introducing a new approach to the detection of small molecules, exemplified here by the direct and selective detection of adenosine monophosphate at the nanomolar scale.

Determination of copy number of short tandem repeat using NAD-dependent ligase and pyrosequencing-compatible method.

A sensitive and pyrosequencing-compatible method for determining the copy number of the short tandem repeat (STR) is presented in this study. When Escherichia coli ligase catalyzes the ligation of primer and probes complementary to the proper sites of the target DNA template, it converts nicotinamide adenine dinucleotide to adenosine monophosphate (AMP) and nicotinamide. The AMP release level is proportional to the copy number of the STR and can be measured using adenylate kinase, pyruvate kinase, and luciferase. Unlike current standard methods based on electrophoresis, the present assay is sensitive to the point mutation. Furthermore, after determination of the copy number of the tandem repeat using the proposed method, the DNA templates, primer and probes immobilized onto super paramagnetic beads can be washed and pyrosequencing can be applied for the remaining DNA sequencing. This assay is specially efficient to handle a large number of samples because massively parallel tests could be executed in a microplate photometer. Furthermore, it can work with the pyrosequencing for further sequencing like genome sequencing.

Nucleotides and nucleosides in ovine and caprine milk during lactation.

The aim of this study was to determine the nucleoside and nucleotide content in ovine and caprine milks at the colostral, transitional, and mature stages of lactation. Samples from 18 dairy sheep and 18 dairy goats were collected at 1, 2, 3, 4, 5, and 15 d postpartum. Separation and quantitation of the 5'-nucleotides (NT) and the nucleosides (NS) was performed by reverse phase HPLC. For each compound measured, considerable interindividual variation was recorded in both species of milk. The total NS content ranged from 57 to 132 micromol/L and from 54 to 119 micromol/L in ovine and caprine milk, respectively. The major NS identified in both species of milk was uridine, representing more than 60% of the total NS pool. The mean levels of inosine and guanosine were comparable between ewe and goat milk. Instead, the mean level of cytidine across the sampling period was much higher in ewe milk (11.9 micromol/L compared with 4.5 micromol/L in goat milk) and exhibited a peak value on the fourth day of lactation. The adenosine content was at least 3-fold higher in caprine milk compared with its ovine counterpart. The total NS and orotic acid contents did not differ significantly between the 2 species. However, in the case of total NT content, interspecies differences were significant, with NT levels ranging from 294 to 441 micromol/L in ovine milk and from 166 to 366 micromol/L in caprine milk. The NT content in colostrum (1-3 d) of both species was higher than in mature milk (15 d), and uridine monophosphate was the dominant NT in all samples.

Umami compounds are a determinant of the flavor of potato (Solanum tuberosum L.).

Vegetable flavor is an important factor in consumer choice but a trait that is difficult to assess quantitatively. The purpose of this study was to assess the levels of the major umami compounds in boiled potato tubers, in cultivars previously assessed for sensory quality. The free levels of the major umami amino acids, glutamate and aspartate, and the 5'-nucleotides, GMP and AMP, were measured in potato samples during the cooking process. Tubers were sampled at several time points during the growing season. The levels of both glutamate and 5'-nucleotides were significantly higher in mature tubers of two Solanum phureja cultivars compared with two Solanum tuberosum cultivars. The equivalent umami concentration was calculated for five cultivars, and there were strong positive correlations with flavor attributes and acceptability scores from a trained evaluation panel, suggesting that umami is an important component of potato flavor.

Nucleotides in sow colostrum and milk at different stages of lactation.

An experiment was conducted with the objective of measuring the concentrations of total milk solids (TMS), CP, and 5'monophosphate nucleotides in sow colostrum and milk. Twelve multiparous sows (Landrace x Yorkshire x Duroc) were used. Litter size was standardized at 11 piglets for all sows at farrowing. Sows were fed an 18.45% CP corn-soybean meal-based diet throughout lactation. The experimental period was the initial 28 d of lactation, with colostrum collected within 12 h of farrowing and milk collected on d 3, 7, 14, 21, and 28. Colostrum and milk samples were analyzed for TMS, CP, adenosine 5'monophosphate (5'AMP), cytidine 5'monophosphate (5'CMP), guanosine 5'monophosphate (5'GMP), inosine 5'monophosphate (5'IMP), and uridine 5'monophosphate (5'UMP). Total milk solids decreased (P < 0.05) from 26.7% on d 0 to 23.1% on d 3. The TMS further decreased (P < 0.05) to 19.3% on d 7, but remained relatively constant thereafter at 18.2, 18.8, and 19.2% on d 14, 21, and 28, respectively. The concentration of CP decreased from 16.6% in colostrum to 7.7, 6.2, 5.5, 5.7, and 6.3% in milk collected on d 3, 7, 14, 21, and 28, respectively (linear and quadratic effect; P < 0.05). Concentrations of 5'AMP, 5'CMP, 5'GMP, and 5'IMP increased from d 0 to d 3 and d 7, and then decreased during the remaining lactation period (quadratic effect; P < 0.05). The concentration of 5'UMP decreased from d 0 to 28 of lactation (linear and quadratic effects; P < 0.05). In colostrum, 5'UMP represented 98% of all 5'monophosphate nucleotides, and in milk, 5'UMP accounted for 86 to 90% of all nucleotides, regardless of day of lactation. The results of this experiment suggest that the concentrations of TMS and CP in sow mammary secretions changed during the first week of lactation, but were constant thereafter. Likewise, the concentrations of 5'monophosphate nucleotides changed during the initial week postpartum, but during the last 2 wk of a 4-wk lactation period, the concentrations were constant.

Glutamine administration during total parenteral nutrition protects liver adenosine nucleotides during and after subsequent hemorrhagic shock.

BACKGROUND: Glutamine supplementation of total parenteral nutrition (TPN) in stressed patients has been advocated. To determine whether glutamine supplementation affects the host response to conditions of stress, animals were given TPN with or without glutamine for 7 days. They were then subjected to the acute stress of hemorrhagic shock, which results in marked loss of hepatic adenosine triphosphate (ATP) and adenosine diphosphate (ADP), with accumulation of adenosine monophosphate (AMP) and the metabolites adenosine, inosine, hypoxanthine, and xanthine. This loss of ATP and accumulation of metabolites contributes to subsequent tissue damage. The hypothesis of the study was that glutamine supplementation would significantly improve restoration of hepatic adenosine nucleotides before and after hemorrhagic shock. METHODS: Sprague-Dawley rats were given TPN for 7 days. One half of the animals (n = 8) received TPN supplemented with glutamine, while one half received TPN with an isonitrogenous mixture of alanine and glycine. Animals were subjected to hemorrhagic shock for 30 minutes and then resuscitated using only heparinized shed blood. Liver biopsies were taken pre- and post-shock, and at 30 and 60 minutes after resuscitation. ATP, ADP, AMP, and their metabolites were measured using gradient high-performance liquid chromatography. RESULTS: After 7 days of TPN, baseline values of ATP, ADP, AMP, and metabolites were similar between the 2 groups before the initiation of shock. Glutamine-treated animals manifested a 40% decrease in ATP level immediately after shock and recovered to 90% of baseline within 60 minutes. By contrast, the control animals manifested a 66% decrease in ATP level after the shock period and recovered only to 60% of baseline at 1 hour postresuscitation. Similar changes were observed in ADP levels and were accompanied by corresponding changes in AMP and adenosine metabolites, all of which rose during shock and fell after resuscitation. CONCLUSIONS: Glutamine supplementation significantly protected the liver from tissue damage caused by hemorrhagic shock. ATP levels remained higher during shock and recovered more rapidly after resuscitation. Glutamine supplementation may help to protect cellular energy stores in the stressed organism and may offer opportunities for therapeutic intervention during and after stress.

Moment analysis as a systematic tool for NMR powder pattern analysis.

The low order moments for chemical shift and second-order quadrupolar powder patterns have been calculated as functions of the anisotropy and asymmetry parameter of the governing interaction, and the expressions inverted to give these parameters as a function of the moments. Theoretical simulations and experimental experience show that moment analysis in most cases equals and in some cases exceeds the accuracy of direct inspection as a method of obtaining NMR parameters. We illustrate the efficacy of the method applied to 31P chemical shift spectra of nucleic acids, and 39K second-order patterns of series of potassium salts.

The effects of halothane and isoflurane on the phosphoenergetic state of the liver during hemorrhagic shock in rats: an in vivo 31P nuclear magnetic resonance spectroscopic study.

We studied the effects of halothane versus isoflurane on the phosphoenergetic state and intracellular pH (pHi) of the rat liver using in vivo 31P nuclear magnetic resonance (NMR) spectroscopy during and after hemorrhagic shock. Seventeen rats were anesthetized with 1 minimum alveolar anesthetic concentration of halothane or isoflurane. The mean arterial blood pressure was reduced to 40 mm Hg and maintained at this level for 45 min by withdrawing blood from the common carotid artery. The shed blood was then returned slowly. In vivo 31P NMR spectra were consecutively collected throughout the study. The phosphoenergetic state of the liver was evaluated from the changes in adenosine triphosphate (ATP) and inorganic phosphate (P(i)) levels. pHi was calculated from the chemical shifts of P(i) and alpha-ATP peaks. During hemorrhagic shock, beta-ATP decreased to 35% and 45%, and P(i) increased to 300% and 230% of their initial values in the halothane and isoflurane groups, respectively. Intracellular acidosis was more severe in the halothane group. The recoveries of beta-ATP and P(i) were better in the isoflurane group. Halothane showed a more detrimental effect than isoflurane on the hepatic phosphoenergetic level during and after hemorrhagic shock.

Phosphorous metabolites in boar spermatozoa. Identification of AMP by multinuclear magnetic resonance.

Boar spermatozoa revealed three prominent resonances in the 31P-NMR spectrum of intact cells. Two of these are known to be GPC and Pi, the third is a phosphomononoester (PME), the identification of which was carried out by proton-detected 2D 1H,31P and 1H,13C chemical shift correlation experiments with gradient selection. The PME was unambiguously assigned to adenosine 5'-monophosphate (AMP). The identification was confirmed by an AMP consuming enzymatic assay. Other physiologically relevant PME's, in particular inosine 5-monophosphate (IMP) and sugar phosphates, were excluded. The intensity of the 31P signal of AMP in boar sperm extract was much higher than those of ADP and ATP, and in intact cells only AMP but no ATP was visible.

Detection and quantitation of phosphorus metabolites in crude tissue extracts by 1H and 31P NMR: use of gradient assisted 1H-31P HMQC experiments, with selective pulses, for the assignment of less abundant metabolites.

The analysis of crude tissue extracts by NMR has proven to be of use in the study of metabolism due to the non-destructive and non-selective character of the technique. Lists of 1H and 31P NMR assignments of phosphorus metabolites in water solution at specified pH and ionic composition are of large general value but their usefulness may be limited when analysing complex mixtures of metabolites at low concentrations. In this work we report on the use of gradient-assisted proton detected multiple quantum 1H and 31P coherence experiments with selective pulses for the rapid and unambiguous assignments of some crowded regions in 1H and 31P spectra of crude extracts from rat liver. The amplitudes of the gradient episodes were calibrated to optimize the coherence transfer pathway between proton and phosphorus, and the delay for the evolution of the long-range coupling was calculated from values of 3JPH and 4JPH ranging from 1.4 to 7.5 Hz. Moreover, a selective 90 degrees Gaussian pulse on the 31P channel was introduced to increase the resolution in the F1-domain and make the method even faster. The procedure was then applied to unambiguously assign the ID 31P and 1H spectra of perchloric acid extracts of rat livers that had been stimulated with phenylephrine, dBcAMP and glucagon and thus detect changes in the concentration of less abundant metabolites such as phosphoenolpyruvate, UDP-glucose and AMP. The fact that the quantification of these metabolites by either 31P and 1H methods lead to different results is discussed, and the use of 1H NMR spectroscopy for the quantification of phosphorus metabolites whose signal are too weak or poorly resolved in a 31P spectrum is proposed.

Human urinary trypsin inhibitor, urinastatin, prevents pancreatic injuries induced by pancreaticobiliary duct obstruction with cerulein stimulation and systemic hypotension in the rat.

OBJECTIVE: The protective effects of human urinary trypsin inhibitor against pancreatic injuries in multifactor-related experimental model of acute pancreatitis were evaluated. DESIGN: Experimental study. MATERIALS AND METHODS: Acute pancreatitis was induced by short-termed (1-hour) pancreatico-biliary duct obstruction with cerulein stimulation (30 minutes; 0.2 microgram/kg per hour) and systemic hypotension (30 minutes; 30% reduction of mean arterial pressure) in rats. In this model, the protective effects of UTI against pancreatic injuries were evaluated at a dose of 10,000 U/kg per hour. RESULTS: In this model, significant increases in portal serum amylase, cathepsin B and malate dehydrogenase levels were observed as compared with the control rats. The redistribution of cathepsin B from the lysosomal to the zymogen fraction and activation of trypsinogen were also observed. Moreover, the increased lysosomal and mitochondrial fragility as well as impaired pancreatic adenylate energy metabolism were noted. The therapeutic administration of human urinary trypsin inhibitor had significant protective effects against these pancreatic injuries. Furthermore, the combined prophylactic and therapeutic administration of human urinary trypsin inhibitor had more significant protective effects than only therapeutic treatment. CONCLUSIONS: These results suggest the importance of timing and of selecting a pertinent protease inhibitor, such as urinary trypsin inhibitor, in the treatment of pancreatitis.

Effect of standard-dose versus high-dose epinephrine on myocardial high-energy phosphates during ventricular fibrillation and closed-chest CPR.

STUDY OBJECTIVE: To evaluate the effects of standard-dose versus high-dose epinephrine on myocardial high-energy phosphate metabolism during resuscitation from cardiac arrest. DESIGN: Prospective, nonrandomized, controlled study using a swine model of cardiac arrest and resuscitation. INTERVENTIONS: After anesthesia, intravascular pressure instrumentation, and ten minutes of ventricular fibrillation arrest, closed-chest CPR was begun. After three minutes of CPR, animals were allocated to receive either 0.02 mg/kg i.v. standard-dose epinephrine (eight) or 0.2 mg/kg i.v. high-dose epinephrine (nine). The animals underwent thoracotomy and rapid-freezing transmural myocardial core biopsy for high-energy phosphate analysis 3.5 minutes after epinephrine administration. High-energy phosphate values were blindly determined using high-pressure liquid chromatography. RESULTS: Intravascular pressure (mm Hg) and high-energy phosphate (nmol/mg protein) results for standard-dose epinephrine versus high-dose epinephrine are, respectively, coronary perfusion pressure, 15.3 +/- 7.8 versus 23.7 +/- 5.5 (P = .0009); phosphocreatine, 0.4 +/- 0.8 versus 6.2 +/- 4.4 (P = .0003); adenosine triphosphate, 9.8 +/- 4.8 versus 12.7 +/- 5.7 (P = .30); adenosine diphosphate, 5.4 +/- 2.1 versus 6.1 +/- 1.3 (P = .41); and adenylate charge, 0.68 +/- 0.12 versus 0.72 +/- 0.12 (P = .87). CONCLUSION: High-dose epinephrine does not deplete myocardial high-energy phosphate when given in this model of prolonged ventricular fibrillation. High-dose epinephrine increases coronary perfusion pressure compared with standard-dose epinephrine. High-dose epinephrine administration repletes phosphocreatine during closed-chest CPR, thereby increasing myocardial energy stores.

Ultrastructural localization of 5'AMP odorant receptor sites on the dendrites of olfactory receptor neurons of the spiny lobster.

A unique probe--biotinylated adenosine-5'-monophosphate (5'AMP-biotin)--was used in transmission electron microscopic (TEM) studies to localize 5'AMP odorant binding sites on the dendrites of olfactory receptor neurons in the aesthetasc sensilla of the spiny lobster, Panulirus argus. This probe is capable of both binding to and exciting 5'AMP-sensitive olfactory receptor neurons, as revealed through biochemical and electrophysiological assays. TEM studies showed that 5'AMP-biotin binding sites are distributed along the entire dendritic region that is exposed to odorants, including the transitional zone (between the inner and outer dendritic segments, including the ciliary segment) and all of the outer dendritic segment. The density of 5'AMP binding sites per micron2 of membrane is similar along the length of the olfactory dendrite. However, the relative number of 5'AMP-biotin binding sites per micron2 of sensillar area diminishes in the distal 30% of the aesthetasc due to a decrease in the amount of dendritic membrane in that region. The distribution of these 5'AMP binding sites is therefore much more extensive than that of enzymes that inactivate 5'AMP--5'ectonucleotidase/phosphatase--which are restricted to the transitional zone (Gleeson et al., 1991). Taken together, these results suggest that 5'AMP-biotin is labeling 5'AMP-specific olfactory receptor sites that are located along the entire outer dendritic segment and that can be coupled to olfactory transduction. This study represents the first in situ localization of specific olfactory receptor sites using a specific, functionally defined ligand.

Different energy metabolism in two human small cell lung cancer subpopulations examined by 31P magnetic resonance spectroscopy and biochemical analysis in vivo and in vitro.

Two human small cell lung cancer tumor lines, maintained as solid tumor xenografts on nude mice and as in vitro cell cultures, were studied by in vivo 31P magnetic resonance spectroscopy and by biochemical analysis of extracts of solid tumors and cell cultures. The tumor lines CPH SCCL 54A and CPH SCCL 54B are subpopulations from the same tumor. In solid tumors (n = 125), the ATP/Pi ratio was greater in 54A than in 54B. This was due to a higher ATP level in 54A, whereas there was no difference in Pi, ADP, and AMP. A decrease in ATP/Pi during growth was caused by a decline in ATP, whereas Pi remained unchanged. Small amounts of phosphocreatine were found in the xenografts and in tumor extracts, but not in the cell extracts; correspondingly, there was a low creatine kinase activity in solid tumors and no activity in the cell cultures. Thus, the phosphocreatine content of the solid tumors originated from the stroma. A difference in ATP content between 54A and 54B was also found in cell cultures; hence, the metabolic difference is an intrinsic quality of the malignant cells and is not caused by the host system.

The cryoprotective effects of dimethyl sulfoxide on human bone marrow as studied by 31P nuclear magnetic resonance spectroscopy.

31P nuclear magnetic resonance spectroscopy has been used to study freshly aspirated normal human bone marrow samples. The pH within the intact cells of the samples was determined from the chemical-shift position of the resonance for inorganic phosphate within the cell; the intracellular pH was found to be 7.35 for fresh bone marrow. The chemical-shift positions of the alpha and beta phosphate resonances of adenosine 5'-triphosphate were used to assess the fraction of this metabolite complexed with Mg2+. It was found that 84% of the total intracellular adenosine 5'-triphosphate was in the Mg2+-complexed form. The concentration of Mg2+ uncomplexed to any ligand was 0.4 mM. The areas of the resonances for the major phosphorus-containing metabolites were used to determine intracellular concentrations. For fresh human bone marrow, the intracellular concentrations determined were phosphate monoesters less than 0.3 mM, 2,3-diphosphoglycerate 3.9 +/- 1.0 mM, inorganic phosphate 1.2 +/- 0.6 mM, phosphodiesters 2.8 +/- 1.0 mM, adenosine 5'-triphosphate 1.6 +/- 0.4 mM, adenosine 5-diphosphate less than 0.2 mM, and nicotinamide adenine dinucleotide less than 0.2 mM. These metabolite concentrations within the intact cell samples did not change over 2.0 h and changed only gradually over a 24-h period. 31P nuclear magnetic resonance spectroscopy was then used to study the cryopreservation of normal human bone marrow in the presence of increasing concentrations of the penetrating cryopreservative dimethyl sulfoxide. Dimethyl sulfoxide alone without freezing was found to cause some gradual hydrolysis of 2,3-diphosphoglycerate, presumably by stimulating diphosphoglycerate phosphatase. The effect of freezing human bone marrow to liquid nitrogen temperatures, storage, and rapid thawing was a dramatic fall in intracellular adenosine 5'-triphosphate levels. The half-life of the metabolite, after thawing, was about 0.3 h. If the bone marrow was frozen in the presence of 2.5, 5.0, and 7.5% dimethyl sulfoxide, the post-thaw half-life was prolonged to 0.4, 0.5, and 0.6 h, respectively. 15% dimethyl sulfoxide afforded complete cryoprotection, with adenosine 5'-triphosphate levels constant for 15 h after thawing the human bone marrow.

The nucleotide profile of human milk.

To further identify and characterize the nitrogen fraction of human milk, nucleotide and total nitrogen contents were determined using high pressure liquid chromatography and Kjeldahl analyses. Five lactating women were followed longitudinally. Each provided 16 milk samples (8-10 ml each) collected before and after a single nursing, and in the morning and afternoon of a single day. This collection scheme was followed at 2, 4, 8, and 12 wk postpartum. The variance pattern of nucleotides was observed to be distinct from that of total nitrogen. As the lactation period progressed from wk 2 to 12 postpartum, levels of cytidine 5' monophosphate and adenosine 5' monophosphate declined from 594 to 321 micrograms/100 ml and from 244 to 143 micrograms/100 ml, respectively, whereas levels of inosine 5' monophosphate increased from 158 to 290 micrograms/100 ml and levels of total nucleotide nitrogen remained constant. Nucleotide nitrogen accounted for approximately 0.1-0.15% of the total nitrogen content of human milk samples analyzed. Total concentration of human milk was observed to decrease as lactation progressed and to be higher in afternoon than in morning samples. The nucleotide profile of human milk was characteristically different from that of other milks commonly used an infant feeding. It is estimated that an infant consuming human milk as a principal nutrition source would ingest 1.4-2.1 mg of nucleotide nitrogen per day.