Major venom proteins of the fire ant Solenopsis invicta: insights into possible pheromone-binding function from mass spectrometric analysis.

Proteins in the venom of the fire ant Solenopsis invicta have been suggested to function in pheromone binding. Venom from queens and workers contains different isoforms of these proteins, consistent with the differing pheromones they secrete, but questions remain about the venom protein composition and glandular source. We found that the queen venom contains a previously uncharacterized pheromone-binding protein paralogue known as Sol i 2X1. Using imaging mass spectrometry, we located the main venom proteins in the poison sac, implying that pheromones might have to compete with venom alkaloids for binding. Using the known structure of the worker venom protein Sol i 2w, we generated three-dimensional homology models of the worker venom protein Sol i 4.02, and of the two main venom proteins in queens and female alates, Sol i 2q and Sol i 2X1. Surprisingly, the models show that the proteins have relatively small internal hydrophobic binding pockets that are blocked by about 10 amino acids of the C-terminal region. For these proteins to function as carriers of hydrophobic ligands, a conformational change would be required to displace the C-terminal region, somewhat like the mechanism known to occur in the silk moth pheromone-binding protein.

Confirmation of the anxiolytic-like effect of dihydrohonokiol following behavioural and biochemical assessments.

Previous studies in this laboratory revealed that dihydrohonokiol-B (DHH-B; 3'-(2 propenyl)-5-propyl-(1,1'-biphenyl)-2,4'-diol), a partially reduced derivative of honokiol, was an effective anxiolytic-like agent in mice at an oral dose of 0.04 mg kg(-1), and at higher doses, when evaluated by the elevated plus-maze test. The aim of this study was to further confirm the anxiolytic-like effect of DHH-B using an additional behavioural procedure (Vogel's conflict test in mice) and a biochemical assessment (in-vitro determination of muscimol-stimulated 36Cl- uptake into mouse cortical synaptoneurosomes). As in earlier experiments, DHH-B (0.04-1 mg kg(-1), p.o.) was shown to prolong the time spent in the open-sided arms of the elevated plus-maze in a dose-dependent manner. Moreover, in the Vogel's conflict test, DHH-B (5 mg kg(-1), p.o.) significantly increased punished water intake. In tests with mouse cerebral cortical synaptoneurosomes, 10 and 30 microM of DHH-B significantly increased 36Cl- influx in the absence of muscimol. In the presence of 25 microM muscimol, the addition of 1 microM DHH-B led to significant enhancement of 36Cl- uptake, while 30 microM DHH-B was required to further stimulate the 36Cl- uptake induced by 250 microM muscimol. The results of these studies confirm that DHH-B is a potent anxiolytic-like agent and that GABA(A) receptor-gated Cl(-)-channel complex is involved in the anxiolytic-like efficacy of DHH-B.

Melatonin directly scavenges hydrogen peroxide: a potentially new metabolic pathway of melatonin biotransformation.

A potential new metabolic pathway of melatonin biotransformation is described in this investigation. Melatonin was found to directly scavenge hydrogen peroxide (H(2)O(2)) to form N(1)-acetyl-N(2)-formyl-5-methoxykynuramine and, thereafter this compound could be enzymatically converted to N(1)-acetyl-5-methoxykynuramine by catalase. The structures of these kynuramines were identified using proton nuclear magnetic resonance, carbon nuclear magnetic resonance, and mass spectrometry. This is the first report to reveal a possible physiological association between melatonin, H(2)O(2), catalase, and kynuramines. Melatonin scavenges H(2)O(2) in a concentration-dependent manner. This reaction appears to exhibit two distinguishable phases. In the rapid reaction phase, the interaction between melatonin and H(2)O(2) reaches equilibrium rapidly (within 5 s). The rate constant for this phase was calculated to be 2.3 x 10(6) M(-1)s(-1). Thereafter, the relative equilibrium of melatonin and H(2)O(2) was sustained for roughly 1 h, at which time the content of H(2)O(2) decreased gradually over a several hour period, identified as the slow reaction phase. These observations suggest that melatonin, a ubiquitously distributed small nonenzymatic molecule, might serve to directly detoxify H(2)O(2) in living organisms. H(2)O(2) and melatonin are present in all subcellular compartments; thus, presumably, one important function of melatonin may be complementary in function to catalase and glutathione peroxidase in keeping intracellular H(2)O(2) concentrations at steady-state levels.

Mass spectrometric analysis of platelet-activating factor after isolation by solid-phase extraction and direct derivatization with pentafluorobenzoic anhydride.

Platelet-activating factor is the term used to denote a class of extremely potent lipid mediators that consist predominantly of 1-O-alkyl- and 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholines. A method has been devised for rapid isolation of these acetylated phospholipids by solid-phase extraction prior to direct derivatization with pentafluorobenzoic anhydride and analysis by gas chromatography (GC)/electron-capture mass spectrometry. Recovery through the entire method (lipid isolation, derivatization, and purification) typically ranged from 70% to 85%. Using the direct derivatization procedure described here, the practical limit of detection for each of the standard alkyl- and acyl-platelet-activating factor homologs was 1 fmol injected into the GC. Results from the application of the method to the analysis of alkyl and acyl homologs of platelet-activating factor isolated from stimulated human umbilical vein endothelial cells are presented, exhibiting excellent accuracy and precision for a wide range of tissue levels of this class of potent autacoids.

Comparative assessment of the anxiolytic-like activities of honokiol and derivatives.

Honokiol has previously been shown to be an effective anxiolytic-like agent in mice when administered for 7 days at 0.2 mg/kg/day prior to evaluation in an elevated plus-maze, while 20 mg/kg is required for efficacy as a single oral dose. The aim of this study was to find analogs of honokiol that are more effective for acute administration. Among the eight analogs evaluated, one partially reduced derivative of honokiol [3'-(2-propenyl)-5-propyl-(1,1'-biphenyl)-2,4'-diol] exhibited significant anxiolytic-like activity at 0.04 mg/kg. Following oral administration of 1 mg/kg of this analog, anxiolytic-like activity was clearly evident at 1 h, peaked at 3 h, and remained significant for longer than 4 h after treatment. Combined administration of the derivative with diazepam led to enhanced anxiolytic-like efficacy. Moreover, as with diazepam, the anxiolytic-like effect of the analog was reduced by flumazenil. In contrast, bicuculline, a GABA(A) antagonist, had no effect on the activity of the derivative. Taken together, these results suggest that this analog of honokiol acts at the benzodiazepine recognition site of the GABA(A)-benzodiazepine receptor complex.

Isolation and identification of a phenolic antioxidant from Aloe barbadensis.

A potent antioxidative compound has been isolated from a methanolic extract of Aloe barbadensis Miller using a combination of column and thin-layer chromatography. The antioxidant activity of this substance was similar to that of alpha-tocopherol as assessed in vitro using rat brain homogenates. On the basis of electrospray ionization and electron-impact ionization mass spectra in combination with reversed-phase, high-performance liquid chromatographic behavior, this compound has been identified as 8-C-beta-D-[2-O-(E)-coumaroyl]glucopyranosyl-2-[2-hydroxy]-propyl-7-methoxy-5-methylchromone.

Strategies for the synthesis of labeled peptides.

Labeled peptides synthesized by core facilities are frequently used by researchers for following trafficking of a peptide, for binding studies, to determine substrate specificity, and for receptor cross-linking studies.The membership of the Association of Biomolecular Resource Facilities was asked to participate in a study focusing on synthesis of a biotin-labeled peptide, and it was suggested that a new strategy, using Rink amide 4-methylbenzhydrylamine resin coupled with Fmoc-Lys(Dde)-OH, be used.This strategy can be used for addition of a variety of labels other than biotin and should prove useful to core facilities. Comparison of the new strategy to other strategies was performed. Biotin labeling has long been assumed to be routine and specific. Despite the assumed routine nature of synthesizing biotinylated peptides, 9 of the 34 samples submitted did not contain any of the correct product. Although synthesis using Fmoc-Lys(Dde)-OH plus biotin generally gave the highest yields, other approaches also yielded a high percentage of the correct product.Therefore, the various strategies are generally comparable. The major advantage of this new approach is that other labels such as fluorescein, dansyl groups, methyl coumarin, and potentially fluorophores and quenchers used for fluorescence resonance energy transfer (FRET) can be directly incorporated into peptides.

Identification of highly elevated levels of melatonin in bone marrow: its origin and significance.

Bone marrow is an important tissue in generation of immunocompetent and peripheral blood cells. The progenitors of hematopoietic cells in bone marrow exhibit continuous proliferation and differentiation and they are highly vulnerable to acute or chronic oxidative stress. In this investigation, highly elevated levels of the antioxidant melatonin were identified in rat bone marrow using immunocytochemistry, radioimmunoassay, high performance liquid chromatography with electrochemical detection and mass spectrometry. Night-time melatonin concentrations (expressed as pg melatonin/mg protein) in the bone marrow of rats were roughly two orders of magnitude higher than those in peripheral blood. Measurement of the activities of the two enzymes (N-acetyltransferase (NAT) and hydroxyindole-O-methoxyltransferase (HIOMT)) which synthesize melatonin from serotonin showed that bone marrow cells have measurable NAT activity, but they have very low levels of HIOMT activity (at the one time they were measured). From these studies we could not definitively determine whether melatonin was produced in bone marrow cells or elsewhere. To investigate the potential pineal origin of bone marrow melatonin, long-term (8-month) pinealectomized rats were used to ascertain if the pineal gland is the primary source of this antioxidant. The bone marrow of pinealectomized rats, however, still exhibited high levels of melatonin. These results indicate that a major portion of the bone marrow's melatonin is of extrapineal origin. Immunocytochemistry clearly showed a positive melatonin reaction intracellularly in bone marrow cells. A melatonin concentrating mechanism in these cells is suggested by these findings and this may involve a specific melatonin binding protein. Since melatonin is an endogenous free radical scavenger and an immune-enhancing agent, the high levels of melatonin in bone marrow cells may provide on-site protection to reduce oxidative damage to these highly vulnerable hematopoietic cells and may enhance the immune capacity of cells such as lymphocytes.

Holmium:YAG laser lithotripsy: A dominant photothermal ablative mechanism with chemical decomposition of urinary calculi.

BACKGROUND AND OBJECTIVE: Evidence is presented that the fragmentation process of long-pulse Holmium:YAG (Ho:YAG) lithotripsy is governed by photothermal decomposition of the calculi rather than photomechanical or photoacoustical mechanisms as is widely thought. The clinical Ho:YAG laser lithotriptor (2.12 microm, 250 micros) operates in the free-running mode, producing pulse durations much longer than the time required for a sound wave to propagate beyond the optical penetration depth of this wavelength in water. Hence, it is unlikely that shock waves are produced during bubble formation. In addition, the vapor bubble induced by this laser is not spherical. Thus the magnitude of the pressure wave produced at cavitation collapse does not contribute significantly to lithotripsy. STUDY DESIGN/MATERIALS AND METHODS: A fast-flash photography setup was used to capture the dynamics of urinary calculus fragmentation at various delay times following the onset of the Ho:YAG laser pulse. These images were concurrently correlated with pressure measurements obtained with a piezoelectric polyvinylidene-fluoride needle-hydrophone. Stone mass-loss measurements for ablation of urinary calculi (1) in air (dehydrated and hydrated) and in water, and (2) at pre-cooled and at room temperatures were compared. Chemical and composition analyses were performed on the ablation products of several types of Ho:YAG laser irradiated urinary calculi, including calcium oxalate monohydrate (COM), calcium hydrogen phosphate dihydrate (CHPD), magnesium ammonium phosphate hexahydrate (MAPH), cystine, and uric acid calculi. RESULTS: When the optical fiber was placed perpendicularly in contact with the surface of the target, fast-flash photography provided visual evidence that ablation occurred approximately 50 micros after the initiation of the Ho:YAG laser pulse (250-350 micros duration; 375-400 mJ per pulse), long before the collapse of the cavitation bubble. The measured peak acoustical pressure upon cavitation collapse was negligible (< 2 bars), indicating that photomechanical forces were not responsible for the observed fragmentation process. When the fiber was placed in parallel to the calculus surface, the pressure peaks occurring at the collapse of the cavitation were on the order of 20 bars, but no fragmentation occurred. Regardless of fiber orientation, no shock waves were recorded at the beginning of bubble formation. Ablation of COM calculi (a total of 150 J; 0.5 J per pulse at an 8-Hz repetition rate) revealed different Ho:YAG efficiencies for dehydrated calculus, hydrated calculus, and submerged calculus. COM and cystine calculi, pre-cooled at -80 degrees C and then placed in water, yielded lower mass-loss during ablation (20 J, 1.0 J per pulse) compared to the mass-loss of calculi at room temperature. Chemical analyses of the ablated calculi revealed products resulting from thermal decomposition. Calcium carbonate was found in samples composed of COM calculi; calcium pyrophosphate was found in CHPD samples; free sulfur and cysteine were discovered in samples composed of cystine samples; and cyanide was found in samples of uric acid calculi. CONCLUSION: These experimental results provide convincing evidence that long-pulse Ho:YAG laser lithotripsy causes chemical decomposition of urinary calculi as a consequence of a dominant photothermal mechanism.

Holmium: YAG lithotripsy: photothermal mechanism.

OBJECTIVE: A series of experiments were conducted to test the hypothesis that the mechanism of holmium:YAG lithotripsy is photothermal. METHODS AND RESULTS: To show that holmium:YAG lithotripsy requires direct absorption of optical energy, stone loss was compared for 150 J Ho:YAG lithotripsy of calcium oxalate monohydrate (COM) stones for hydrated stones irradiated in water (17+/-3 mg) and hydrated stones irradiated in air (25+/-9 mg) v dehydrated stones irradiated in air (40+/-12 mg) (P < 0.001). To show that Ho:YAG lithotripsy occurs prior to vapor bubble collapse, the dynamics of lithotripsy in water and vapor bubble formation were documented with video flash photography. Holmium:YAG lithotripsy began at 60 microsec, prior to vapor bubble collapse. To show that Ho:YAG lithotripsy is fundamentally related to stone temperature, cystine, and COM mass loss was compared for stones initially at room temperature (approximately 23 degrees C) v frozen stones ablated within 2 minutes after removal from the freezer. Cystine and COM mass losses were greater for stones starting at room temperature than cold (P < or = 0.05). To show that Ho:YAG lithotripsy involves a thermochemical reaction, composition analysis was done before and after lithotripsy. Postlithotripsy, COM yielded calcium carbonate; cystine yielded cysteine and free sulfur; calcium hydrogen phosphate dihydrate yielded calcium pyrophosphate; magnesium ammonium phosphate yielded ammonium carbonate and magnesium carbonate; and uric acid yielded cyanide. To show that Ho:YAG lithotripsy does not create significant shockwaves, pressure transients were measured during lithotripsy using needle hydrophones. Peak pressures were <2 bars. CONCLUSION: The primary mechanism of Ho:YAG lithotripsy is photothermal. There are no significant photoacoustic effects.

Study on the synthesis and characterization of peptides containing phosphorylated tyrosine.

Phosphorylation and dephosphorylation are key events in receptor-mediated and post-receptor-mediated signal transduction. Synthetic phosphopeptides have been shown to have dramatic agonist or antagonist effects in several of these signaling pathways. For its 1997 study, the Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group assessed the ability of member laboratories to synthesize phosphotyrosine peptides. Participating laboratories were requested to synthesize and submit the following crude peptide, H-Glu-Asp-Tyr-Glu-Tyr(PO3H2)-Thr-Ala-Arg-Phe-NH2, for evaluation by amino acid analysis, sequence analysis, RP-HPLC, MALDI-TOF and ESI mass spectrometry. Prior to analysis of submitted peptides from ABRF members, the Peptide Synthesis Research Group synthesized and characterized the nonphosphorylated form of the peptide, the doubly phosphorylated form and the peptides singly phosphorylated on either the first or the second tyrosine. These peptide standards were separated easily by HPLC and capillary electrophoresis and the phosphotyrosine was detected readily by Edman degradation sequence analysis. No differences were seen by amino acid analysis and the expected masses were observed by mass spectrometry. The two singly phosphorylated peptides were easily distinguished by MALDI-PSD. Analysis of the peptides submitted from member facilities revealed that all but four of the 33 samples contained the correct product as determined by HPLC and mass spectrometry. HPLC analysis indicated that 20 of the 33 submitted samples contained greater than 75% correct product, five contained less than 50% correct product and four did not contain any correct product. By ESI/MS, an additional singly charged ion at m/z 535.5 was detected in five of the 33 submitted samples; this ion was subsequently shown to represent Ac-TARF-NH2. No correlation was found to exist between coupling time and percentage correct product; however, a correlation may exist between a greater percentage of correct product and the use of non-protected phosphotyrosine.

Identification of magnolol and honokiol as anxiolytic agents in extracts of saiboku-to, an oriental herbal medicine.

The principal active anxiolytic components in Saiboku-to, an Oriental herbal medicine, have been isolated and identified as magnolol (5,5'-di-2-propenyl-1,1'-biphenyl-2,2'-diol) and honokiol (3',5-di-2-propenyl-1,1'-biphenyl-2,4'-diol). Evaluation by means of an elevated plus-maze test showed that honokiol was at least 5000 times more potent than Saiboku-to when mice were treated orally for 7 days.

A novel melatonin metabolite, cyclic 3-hydroxymelatonin: a biomarker of in vivo hydroxyl radical generation.

In the current study, we characterized a urinary melatonin metabolite which could provide a safe and effective method to monitor generation of HO* in humans. Using mass spectrometry (MS), proton nuclear magnetic resonance (1H NMR), COSY 1H NMR analysis, and calculations on the relative thermodynamic stability, a novel melatonin metabolite was identified as cyclic 3-hydroxymelatonin (3-OHM). 3-OHM is the product of the reaction of melatonin with HO* which was generated in two different cell-free in vitro systems. Interestingly, this same metabolite, 3-OHM, was also identified in the urine of both rats and humans. A proposed reaction pathway suggests that 3-OHM is the footprint product that results when a melatonin molecule scavenges two HO*. When rats were challenged with ionizing radiation which results in HO* generation, urinary 3-OHM increased dramatically compared to that of controls. These results strongly indicate that the quantity of 3-OHM produced is associated with in vivo HO* generation. Since melatonin exists in virtually all animal species and has a wide intracellular distribution and 3-OHM is readily detected noninvasively in urine, we suggest that 3-OHM is a valuable biomarker that can be used to monitor in vivo HO* levels in humans and other species. The measurement of urinary 3-OHM as a biomarker of HO* generation could provide clinical benefits in the diagnosis and treatment of diseases.

Quantitative analysis of exogenous peptides in plasma using immobilized enzyme cleavage and gas chromatography-mass spectrometry with negative ion chemical ionization.

A method is presented for the analysis of peptides in plasma at picomole to femtomole levels. Peptides are isolated from plasma by solid-phase extraction, the peptide of interest is purified by reversed-phase high-performance liquid chromatography (HPLC) and selectively digested using immobilized trypsin or chymotrypsin to yield specific di- or tripeptides. These di- and tripeptides are esterified using heptafluorobutyric anhydride, alkylated with pentafluorobenzyl bromide, then quantified by gas chromatography-mass spectrometry with negative ion chemical ionization. This method has been evaluated for a model synthetic heptapeptide, using a deuterium labeled analog as an internal standard. The half-life of the heptapeptide in human plasma was found to be 2 min. Extraction efficiencies of a tritiated peptide of similar size to the heptapeptide, [3H]DSLET, from plasma using either C18 or strong cation-exchange columns were 85+/-3 and 70+/-2%, respectively. Quantitation of fragments from the heptapeptide indicated that the analysis was linear from 1-50 ng of the heptapeptide per ml of plasma. This method was subsequently employed for pharmacokinetic studies of the biologically active peptide Met-enkephalin-Arg-Gly-Leu, where linearity was obtained from 50 to 1000 ng/ml in rat plasma. This method demonstrated negligible side reaction by-products due to autolysis, and has potential for extensive use given the wide availability of gas chromatography-mass spectrometry.

Quantitative analysis of two opioid peptides in plasma by liquid chromatography-electrospray ionization tandem mass spectrometry.

Quantitative analysis of two opioid peptides, DSLET [(D-Ser2)Leu-enkephalin-Thr6] and Met-enkephalin-Arg-Gly-Leu, was performed using microbore liquid chromatography interfaced to electrospray ionization tandem mass spectrometry. Validation of the methodology was demonstrated for each peptide in plasma. Quantitative analyses were performed through the use of a deuterium labelled peptide analog as an internal standard. Linearity was observed for the analysis of DSLET (5-1000 ng/ml) and Met-enkephalin-Arg-Gly-Leu (1-1000 ng/ml) in plasma with a limit of detection of 0.25 ng/ml for Met-enkephalin-Arg-Gly-Leu and 1.0 ng/ml for DSLET. In general, the observed concentrations showed good reproducibility with coefficients of variation of within 15%. In the concentration range studied, only 0.5 ml of plasma was required for optimal detection of Met-enkephalin-Arg-Gly-Leu and 0.25 ml for DSLET. Application of this method was demonstrated by studying the disposition of DSLET in a rat. DSLET administered to a rat exhibited a short half-life and a high clearance value.

Acetylcholine: oscillation of levels in mouse brain following electroshock.

The acetylcholine contents of mouse brain regions were measured in order to investigate the response of cholinergic neurons to electroshock. In this study, mice were subjected to electroshock and then sacrificed by microwave irradiation at time intervals of from 0.4 to 6.9 a after electroshock. In all of the brain regions studied, the acetylcholine concentration appeared to oscillate with a mean period of approximately 2.5 a following electroshock. The rate of recovery of acetylcholine after electroshock was calculated for each brain region from the oscillatory equation obtained by nonlinear regression analysis of the experimental data. The rates of synthesis of acetylcholine derived from these in vivo measurements were substantially higher than have been indicated by other methods.

Schizophrenia, psychosis, and cerebral spinal fluid homovanillic acid concentrations.

Neuroleptic drugs block brain dopamine receptors and are effective in treating psychoses of diverse origins. This finding has become a cornerstone of the dopamine theory of schizophrenia, but clinical studies relating schizophrenia, per se, to brain dopamine metabolism have ranged from controversial to negative. This article presents new evidence that cerebrospinal fluid levels of the dopamine metabolite homovanillic acid are related to the severity of psychosis in schizophrenia. These results support the concept that homovanillic acid levels in cerebrospinal fluid vary as a function of psychosis rather than being related to the diagnosis of schizophrenia per se.

Antiinflammatory C-glucosyl chromone from Aloe barbadensis.

A new antiinflammatory agent identified as 8-[C-beta-D-[2-O-(E)-cinnamoyl]glucopyranosyl]-2- [(R)-2-hydroxypropyl]-7-methoxy-5-methylchromone (1) has been isolated from Aloe barbadensis Miller. At a dose of 200 microg/mouse ear, 1 exhibited topical antiinflammatory activity equivalent to 200 microg/ear of hydrocortisone. There was no reduction in thymus weight caused by treatment with 1 for any of the doses tested, while 200 microg/ear of hydrocortisone resulted in a 50% decrease in thymus weight.

Platelet activating factor and lyso-phosphatidylcholines from strawberry.

Lipids from strawberry fruits, leaves, achenes and pollen were separated into classes by TLC, purified by HPLC and tested for biological activity. A lipid fraction from fruits with the same chromatographic behaviour as authentic platelet activating factor (PAF) showed identical biological activity, namely, dose-dependent aggregation of washed rabbit platelets, inhibition of aggregation by CV 3988, platelet desensitization to PAF and vice versa, and loss of activity by alkaline hydrolysis and recovery of activity by reacetylation. The presence of PAF was confirmed by FAB mass spectrometry. Lyso-phosphatidylcholines, including lyso-PAF, were also found in all the plant parts tested.