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At least 1,009 records · Page 56Linked to original sources
PubMed2003

Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive biodegradable system for paclitaxel delivery.

The main objective of this study was to develop and characterize a pH-sensitive biodegradable polymeric nanoparticulate system for tumor-selective paclitaxel delivery. A representative hydrophobic poly(beta-amino ester) (poly-1) was synthesized by conjugate addition of 4,4'-trimethyldipiperidine with 1,4-butanediol diacrylate. Poly-1 (M(n) 10,000 daltons) nanoparticles were prepared by the controlled solvent displacement method in an ethanol-water system in the presence of Pluronic) F-108, a poly(ethylene oxide) (PEO)-containing non-ionic surfactant. Control and PEO-modified nanoparticles were characterized by Coulter counter, scanning electron microscopy (SEM), zeta potential measurements, and electron spectroscopy for chemical analysis (ESCA). Polymer degradation studies were performed in phosphate-buffered saline (PBS, pH 7.4) at 37 degrees C. Paclitaxel loading capacities and efficiencies were determined and release studies were performed in Tween)-80 (0.1%, w/v)-containing PBS at 37 degrees C. Control and PEO-modified nanoparticles, labeled with rhodamine-123, were incubated with BT-20 cells to examine the uptake and cellular distribution as a function of time. PEO-modified nanoparticles with an average size of 100-150 nm and a positive surface charge of 37.0 mV were prepared. SEM analysis showed distinct smooth, spherical particles. The ether (-C-O-) peak of the C(1s) envelope in ESCA confirmed the surface presence of PEO chains. Polymer biodegradation studies showed that almost 85% of the starting material degraded after 6 days. The maximum paclitaxel loading efficiency attained was 97% at 1.0% (w/w) of the drug. Paclitaxel release studies showed that approximately 10% was released in the first 24 h, 80% after 3 days, and the entire content was released in approximately 5 days. After 1 h of incubation, a large fraction of the administered control and PEO-modified poly-1 nanoparticles was internalized in BT-20 cells. Results of this study demonstrate that PEO-modified poly-1 nanoparticles could provide increased therapeutic benefit by delivering the encapsulated drug to solid tumors.

Drug Delivery Systems↗
PubMed2000

One- and three-year prospective outcome study of modified condylotomy for treatment of reducing disc displacement.

PURPOSE: This outcomes study was designed to provide a comprehensive evaluation of modified condylotomy for the treatment of the painful temporomandibular joint with reducing disc displacement (Wilkes stage II, early III). PATIENTS AND METHODS: A prospective study of 48 consecutive patients (79 joints) was conducted. All reducing disc displacements were verified by disc imaging. Independent evaluations were performed to assess pain, dysfunction, and progression of disease before modified condylotomy and at intervals up to 1 year after operation. Thirty-one patients (50 joints) completed the examination before the operation and 1 year later. Patient-based assessments were made for pain and diet in 22 patients (35 joints) 3 years after operation. RESULTS: The mean (+/- SE) visual analog scale (VAS) score for pain improved from 6.9+/-0.4 before modified condylotomy to 2.0+/-0.4 1 year later (P < .001). Serious pain (VAS score greater than 4) after operation was 7 times more likely (P < .04) when there was persistent disc displacement. The mean frequency of pain each day decreased from 14.6+/-1.4 hours to 4.8+/-1.3 hours (P < .001). Dietary restrictions improved from a mean VAS score of 6.1+/-0.5 before operation to 8.8+/-0.3 at 1 year (P < .001). Small differences between mean VAS scores for pain at 1 (2.0+/-0.5) and 3 (2.7+/-0.5) years and diet at 1 (8.6+/-0.4) and 3 (8.4+/-0.5) years after operation were not significant. Mean maximal incisal opening was 41.7+/-1.2 mm before operation and 43.5+/-1.1 mm 1 year later, but the difference was not statistically significant. Mean contralateral movement improved from 8.1+/-0.3 mm to 8.9+/-0.3 mm 1 year after operation (P < .05). Clicking was reduced from 64% of joints to 16% 1 year after operation (P < .001). The disc was reduced in 72% of joints, healing of an incipient degenerative lesion occurred in 1 joint, and there was no evidence of progression to nonreducing disc displacement (Wilkes late III, IV, V) or DJD (Wilkes IV, V) in any joint 1 year after modified condylotomy. The rate for reoperation was 4%. Complications occurred in 4 patients after operation and were resolved 1 year later. When these outcomes were judged by 7 AAOMS assessment indices for internal derangement, the mean rate of favorable outcome was 94%. CONCLUSION: Modified condylotomy is an effective operation for treating pain and diminished function of temporomandibular joints with reducing disc displacement. It is also an effective treatment for slowing and, in some cases, reversing the progression of internal derangement.

Adolescent↗
PubMed2002

The position of the spine in the recovery position--an experimental comparison between the lateral recovery position and the modified HAINES position.

UNLABELLED: The lateral recovery position is widely used for the positioning of unconscious patients. Ideally, in the setting of trauma it is avoided because of concerns about spinal cord injury. However, unconscious individuals with unsuspected trauma or trauma victims attended by partially trained first-aiders may be placed in the recovery position, potentially endangering the cord. Excessive movement of the spine in the recovery position may increase the risk of spinal cord injury in these situations. A new recovery position, termed the modified HAINES position, is described and the position of the spine in this position is compared with the lateral recovery position. HYPOTHESIS: That the modified HAINES position results in less distortion of the position of the spine than the lateral recovery position. METHODS: Thirty-eight healthy volunteers were imaged in the two different positions. Measurements of rotation, flexion and lateral flexion of the cervical and thoraco-lumbar spine were made. Two tailed paired t-tests were employed to compare measurements of the two positions and a McNemar test was used to compare the subjects' subjective experiences. RESULTS: The modified HAINES position resulted in 13.0 degrees (99% CI: 7.5-18.5) less lateral flexion and 12.6 degrees (99% CI: 9.4-15.9) less extension of the cervical spine while the position of the thoraco-lumbar spine was similar in both positions. Nineteen of 28 subjects found the modified HAINES position more comfortable (not significant). CONCLUSION: The modified HAINES position results in a more neutral position of the spine making it preferable to the lateral recovery position in the management of patients when trauma may have occurred. Further research is required to ensure that the recovery positions in use today are the best possible.

Adolescent↗
PubMed1998

A comparison of standard and a modified method of two resuscitator adult cardiopulmonary resuscitation: description of a new system for research into advanced life support skills.

The study compares two methods of Advanced Life Support by a pair of resuscitators using a bag-valve-mask (BVM) technique. Standard two resuscitator cardiopulmonary resuscitation (CPR) was compared with a modified method of two resuscitator CPR. During the modified CPR one resuscitator held the face mask while the other resuscitator alternates between squeezing the self inflating bag and performing simulated cardiac compressions. Standard CPR was performed at a ventilation:compression ratio of 1:5 while modified CPR was performed at a ventilation:compression ratio of 2:15. Comparisons were made during induction of anaesthesia in 30 ASA I and II patients. Modified CPR produced a greater tidal volume (TV) (P < 0.001), a slower respiratory rate (RR) (P < 0.001) and a faster compression rate (CR) (P < 0.01) (means with (S.D.): modified CPR: TV 990 (220) ml, RR 6 (1) min(-1), CR 82 (8) min(-1); standard CPR: TV 570 (190) ml, RR 10 (2) min(-1), CR 65 (11) min(-1)). A new method for the simultaneous computerised recording of simulated cardiac compressions together with mask pressure and expired gas composition in anaesthetised patients is described.

Adult↗
PubMed2002

Degradation of carbon tetrachloride by modified Fenton's reagent.

The degradation of tetrachloromethane (carbon tetrachloride-CT) by modified Fenton's reagent (catalyzed hydrogen peroxide) was investigated using a range of hydrogen peroxide concentrations and 1 mM iron(III) catalyst. The documented reactive species in modified Fenton's reactions, hydroxyl radical (OH*), is not reactive with CT, yet CT degradation was observed in the Fenton's reactions and was confirmed by chloride generation. Because CT is not reactive with OH*, a reductive mechanism which may involve superoxide radical anion is proposed for CT degradation in modified Fenton's systems. Scavenging of reductants by excess chloroform prevented CT degradation, confirming a reductive mechanism. Similar to CT, three other oxidized aliphatic compounds, hexachloroethane, bromotrichloromethane, and tetranitromethane, were also degraded by modified Fenton's reagent. The results show that modified Fenton's reactions act through a reductive mechanism to degrade compounds that are not reactive with OH*, which broadens the scope of this process for hazardous waste treatment and remediation.

Carbon Tetrachloride↗
PubMed2000

A modified method of planning and delivery for dynamic multileaf collimator intensity-modulated radiation therapy.

PURPOSE: To develop a modified planning and delivery technique that reduces dose nonuniformity for tomographic delivery of intensity-modulated radiation therapy (IMRT). METHODS AND MATERIALS: The NOMOS-CORVUS system delivers IMRT in a tomographic paradigm. This type of delivery is prone to create multiple dose nonuniformity regions at the arc abutment regions. The modified technique was based on the cyclical behavior of arc positions as a function of a target length. With the modified technique, two plans are developed for the same patient, one with the original target and the second with a slightly increased target length and the abutment regions shifted by approximately 5 mm compared to the first plan. Each plan is designed to deliver half of the target prescription dose delivered on alternate days, resulting in periodic shifts of abutment regions. This method was experimentally tested in phantoms with and without intentionally introduced errors in couch indexing. RESULTS: With the modified technique, the degree of dose nonuniformity was reduced. For example, with 1 mm error in couch indexing, the degree of dose nonuniformity changed from approximately 25% to approximately 12%. CONCLUSION: Use of the modified technique reduces dose nonuniformity due to periodic shifts of abutment regions during treatment delivery.

Particle Accelerators↗
PubMed1993

A modified direct technique versus conventional direct placement of brackets: in vitro bond strength comparison.

Bond strength and failure location were evaluated in vitro for two methods of direct bracket bonding. Sixty human premolars were divided into two groups of 30 each. In group I the brackets were bonded with a two-paste adhesive by using the conventional direct method. In group II brackets were bonded with a newly developed modified direct technique. During the modified direct technique, unfilled resin catalyst liquid was applied to a bracket, which had a coating of hardened composite cured against a dental anatomic matrix (tooth). After the acid-etched tooth was coated with unfilled resin base liquid, the bracket was placed. Mixed unfilled resin liquid (sealant) was then placed at the periphery of the bracket/tooth interface. Thus the major modification of the direct technique would entail fabrication by the manufacturer of a bracket with prehardened bis-GMA composite resin on its backing. Bond strengths were 155.2 (SD = 35.7) and 140.6 (SD = 30.1) kg/cm2 for conventional and modified techniques, respectively. With the conventional method, failure occurred mainly at the tooth/composite interface. Failure seen with the modified technique was mixed, but the major mode was composite/bracket. Therefore this modified bonding method promises similar bond strengths and some advantages over the conventional method including, elimination of composite flash from around the brackets, ample working time, consistent adhesive thickness, and reduction of porosity.

Bisphenol A-Glycidyl Methacrylate↗
PubMed1997

Generation of a polyclonal antibody against lipid peroxide-modified proteins.

A specific polyclonal antibody against the lipid peroxide (LOOH)-modified rabbit serum albumin (RSA) was generated in rabbits. The antibody selectively recognized the modified protein in a concentration-dependent manner and did not cross react with aldehyde-modified proteins or proteins directly oxidized with the free radical generator 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). Oxidized low-density lipoprotein (Ox-LDL), but not native LDL, was also recognized by the antibody in a concentration-dependent manner. The antibody also cross reacted with several other proteins modified by LOOH suggesting that the antibody is directed towards a common epitope and not towards the protein sequence. Western blot analysis of normal human plasma showed that at least three different proteins are recognized by the antibody. RAW cells, preincubated with LOOH, were immunostained with the antibody and the antigenic epitopes were present intracellularly, while controls lacking in the primary antibodies failed to show immunoreactivity. Atherosclerotic arteries from cholesterol-fed monkeys and human atherosclerotic lesions were also immunostained by the antibody. The immunoreactivity was co-localized in areas rich in foam cell macrophages. These results suggest that LOOH-modified proteins present an unique antigenic epitope that may represent a primary product of interaction of LOOH with proteins.

Aldehydes↗
PubMed1998

Laboratory evaluation of single-lumen, dual-orifice combined spinal-epidural needles: effects of bevel orientation and modified technique.

STUDY OBJECTIVES: To evaluate the success rate of the spinal needle exiting through the spinal needle orifice in two commonly available single-lumen, dual-orifice combined spinal-epidural (CSE) needle kits, and to study the effects of the epidural needle bevel orientation and the modified insertion technique on its success rate. DESIGN: Prospective, randomized study. SETTING: Laboratory. EQUIPMENT: Two types of single-lumen, dual-orifice CSE needle kits: CSE-Q kit and CSE-S kit. INTERVENTION: 20 anesthesiologists and nurse-anesthetists were assigned to select randomly from the two types of CSE kits (CSE-Q and CSE-S). The epidural needle was then placed into a foam device simulating a patient's back and the associated spinal needle was inserted through the epidural needle with the epidural needle bevel oriented randomly upward, downward, left, and right, respectively. The procedures were performed before and after the modified technique was used. The modified technique consisted of (1) aligning the orifice/bevel of the spinal needle in the same direction as the epidural needle bevel, and (2) gently bending the hub of the spinal needle in the direction of the epidural needle bevel while advancing the spinal needle. MEASUREMENTS AND MAIN RESULTS: Success rate of the spinal needle exiting through the spinal needle orifice was measured. Data were collected from 1600 attempts. The modified technique improved the success rate of spinal needle exiting through the spinal needle orifice from 67% to 94 % for the CSE-Q kit and 50% to 81% for the CSE-S kit. Upward orientation of the epidural needle bevel further improved the success rate to 96% and 91% for CSE-Q and CSE-S kits, respectively. CONCLUSIONS: The spinal needle did not always exit through the spinal needle orifice in the CSE kits tested. CSE-Q kits performed better than CSE-S kits. Under the study conditions, the modified technique and the upward orientation of epidural needle bevel significantly improved the success rate of the spinal needle exiting through the appropriate spinal needle orifice.

Anesthesia, Epidural↗
PubMed2001

Improvement of the physical properties of reprocessed paper by using biological treatment with modified cellulase.

A primary need for waste paper reprocessing is to preserve optical properties and the physical strength of the paper fibers. In this study, modified cellulase with copolymer, polyethylene oxide (PEO) derivatives and maleic anhydride (MA) was applied to the reprocessing of mixed office waste (MOW). Modified cellulase was prepared by a chemical reaction between amino groups of the cellulase and the MA functional groups of the copolymer. In MOW reprocessing, modified cellulase improved several physical properties of the paper including freeness, optical properties, and physical strength compared to the conventional process. Even though native cellulase improved the physical properties, paper treated with modified cellulase exhibited an increase in physical properties such as tensile strength and internal bond over those of unmodified cellulase. From these results, modified cellulase method is a new biological treatment that will save pulp resources, which are added to waste paper reprocessing to maintain the strength of paper.

Cellulase↗
PubMed2003

The use of chemically modified and unmodified cassava waste for the removal of Cd, Cu and Zn ions from aqueous solution.

The use of different chemically modified cassava waste biomass for the enhancement of the adsorption of three metal ions Cd, Cu and Zn from aqueous solution is reported in this paper. Treating with different concentrations of thioglycollic acid modified the cassava waste biomass. The sorption rates of the three metals were 0.2303 min(-1) (Cd(2+)), 0.0051 min(-1) (Cu(2+)), 0.0040 min(-1) (Zn(2+)) and 0.109 min(-1) (Cd(2+)), 0.0069 min(-1) (Cu(2+)), 0.0367 min(-1) (Zn(2+)) for 0.5 and 1.00 M chemically modified levels, respectively. The adsorption rates were quite rapid and within 20-30 min of mixing, about 60-80% of these ions were removed from the solutions by the biomass and that chemically modifying the binding groups in the biomass enhanced its adsorption capacity towards the three metals. The results further showed that increased concentration of modifying reagent led to increased incorporation, or availability of more binding groups, in the biomass matrix, resulting in improved adsorptivity of the cassava waste biomass. The binding capacity study showed that the cassava waste, which is a serious environmental nuisance, due to foul odour released during decomposition, has the ability to adsorb trace metals from solutions.

Adsorption↗
PubMed2001

Immobilisation on polystyrene of diazirine derivatives of mono- and disaccharides: biological activities of modified surfaces.

The potential of surface glycoengineering for biomaterials and biosensors originates from the importance of carbohydrate-protein interactions in biological systems. The strategy employed here utilises carbene generated by illumination of diazirine to achieve covalent bonding of carbohydrates. Here, we describe the synthesis of an aryl diazirine containing a disaccharide (lactose). Surface analysis techniques [X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS)] demonstrate its successful surface immobilisation on polystyrene (PS). Results are compared to those previously obtained with an aryl diazirine containing a monosaccharide (galactose). The biological activity of galactose- or lactose-modified PS samples is studied using rat hepatocytes, Allo A lectin and solid-phase semi-synthesis with alpha-2,6-sialyltransferase. Allo A shows some binding to galactose-modified PS but none to lactose-modified surfaces. Similar results are obtained with rat hepatocytes. In contrast, sialylation of lactose-modified PS is achieved but not with galactose-modified surfaces. The different responses indicate that the biological activity depends not only on the carbohydrate per se but also on the structure and length of the spacer.

Animals↗
PubMed1998

Hypothermic preservation of isolated rat lungs in modified bicarbonate buffer, EuroCollins solution or St Thomas' Hospital cardioplegic solution.

OBJECTIVES: Inadequate preservation solutions limit lung storage times and, consequently, transplant programs. To address this problem we established an isolated, ventilated and perfused rat lung preparation. Here we report the effects of hypothermic storage in EuroCollins solution, St Thomas' Hospital cardioplegic solution and a modified bicarbonate buffer solution. METHODS: Lungs from male Wistar rats (230-330 g) were perfused via the pulmonary artery with modified bicarbonate buffer (37 degrees C, 15 ml/min, constant flow) and ventilated by positive pressure (tidal volume:1.6-1.8 ml, 80 breaths/min). Vascular resistance (pulmonary artery pressure:perfusate flow ratio) and airways compliance (tidal volume:tracheal pressure ratio) were measured. After a control perfusion period (20 min), lungs were flushed with, then immersed in, bicarbonate buffer (4 degrees C) for varying periods (0-24 h). After storage, lung function was assessed during 20 min reperfusion. Having established a suitable period for study, storage in EuroCollins, St Thomas' Hospital cardioplegic solution or bicarbonate buffer were compared. RESULTS: Pulmonary compliance (ml/cmH2O) was significantly (P < 0.05) reduced in lungs stored for 6 h in modified bicarbonate buffer (0.026 +/- 0.008), EuroCollins solution (0.013 +/- 0.002) or St Thomas' Hospital solution (0.025 +/- 0.005) compared to unstored lungs (0.068 +/- 0.007). Vascular resistance, (1.32 +/- 0.13 cmH2O/ml per min) in unstored lungs, was similar in lungs stored in St Thomas' Hospital solution but increased significantly in lungs stored in modified bicarbonate buffer (3.22 +/- 0.78 cmH2O/ml per min) or EuroCollins solution (4.66 +/- 0.57 cmH2O/ml per min). CONCLUSIONS: Hypothermic storage of rat lungs for 6 h in modified bicarbonate buffer or St Thomas' Hospital solution causes less increase in vascular resistance on reperfusion than EuroCollins solution.

Animals↗
PubMed1999

Probing interactions between HIV-1 reverse transcriptase and its DNA substrate with backbone-modified nucleotides.

BACKGROUND: To gain a molecular understanding of a biochemical process, the crystal structure of enzymes that catalyze the reactions involved is extremely helpful. Often the question arises whether conformations obtained in this way appropriately reflect the reactivity of enzymes, however. Rates that characterize transitions are therefore compulsory experiments for the elucidation of the reaction mechanism. Such experiments have been performed for the reverse transcriptase of the type 1 human immunodeficiency virus (HIV-1 RT). RESULTS: We have developed a methodology to monitor the interplay between HIV-1 RT and its DNA substrate. To probe the protein-DNA interactions, the sugar backbone of one nucleotide was modified by a substituent that influenced the efficiency of the chain elongation in a characteristic way. We found that strand elongation after incorporation of the modified nucleotide follows a discontinuous efficiency for the first four nucleotides. The reaction efficiencies could be correlated with the distance between the sugar substituent and the enzyme. The model was confirmed by kinetic experiments with HIV-1 RT mutants. CONCLUSIONS: Experiments with HIV-1 RT demonstrate that strand-elongation efficiency using a modified nucleotide correlates well with distances between the DNA substrate and the enzyme. The functional group at the modified nucleotides acts as an 'antenna' for steric interactions that changes the optimal transition state. Kinetic experiments in combination with backbone-modified nucleotides can therefore be used to gain structural information about reverse transcriptases and DNA polymerases.

Acetylation↗
PubMed2003

Activation of protein kinase C by phorbol esters in human macrophages reduces the metabolism of modified LDL by down-regulation of scavenger receptor activity.

Atherogenesis and inflammation are dependent on macrophage function. Signalling pathways are involved in the modulation of the classical low density lipopotein (LDL)-receptor and scavenger receptors activities, which are both expressed by macrophages. This study has evaluated the role of activation of the protein kinase A and C pathways in human macrophages on the metabolism of lipid carried by native, acetylated and oxidised LDL. We found that [3H]oleate incorporation into cholesteryl ester and triacylglycerol is increased by an analogue of cAMP, but strongly inhibited by treatment with phorbol ester (PMA) (100 nM, 6 h) in the presence of acLDL and oxLDL and, to a lesser extent, nLDL. The mechanisms underlying the effects of the phorbol ester were investigated further. The protein kinase C inhibitors, calphostin C and herbimycin A, prevented the PMA-mediated inhibition of cholesterol esterification. PMA also reduced [14C]acetate incorporation into newly synthesised lipids especially in the presence of nLDL, and reduced the uptake of cholesterol carried by modified LDL. Furthermore, the effects of PMA were not modified by inhibition of proteases activities, ruling out the hypothesis that CD163, a scavenger receptor which is shed by the cell surface in the presence of phorbol, is involved in the phorbol-induced reduction of cholesterol accumulation in macrophages in response to LDL. We conclude that binding of modified LDL to macrophages induces an appropriate pattern of scavenger receptor phosphorylation which, in turn, determines the optimal receptor internalisation process. PMA activates PKC pathways and prevents the optimal ligand-induced phosphorylation of the receptors, compromising the processes of degradation of modified LDL. The data also suggest that this mechanism may be related to the decreased uptake by activated macrophages of lipid carried by modified lipoproteins during the early phases of inflammation (284).

Cholesterol↗
PubMed1998

Immunochemical detection of oxidative DNA damage in cancer and aging using anti-reactive oxygen species modified DNA monoclonal antibody.

The formation of reactive oxygen species (ROS), although a normal cellular activity, is considerably enhanced under chronic inflammatory conditions and ischemia. These species have been implicated in various disorders, mutagenesis, carcinogenesis and aging. Of many macromolecules, DNA is the most susceptible to hydroxyl radical, the most reactive of the ROS. The present study is designed to detect oxidative DNA damage in cancer patients and healthy aged humans using an anti-ROS-DNA monoclonal antibody (mAb). Purified calf thymus DNA fragments (approximate size 400 bp) were modified with OH, generated by UV-irradiation (254 nm) of hydrogen peroxide. ROS-modified DNA was characterized by UV-spectroscopy, melting temperature, alkaline sucrose density gradient ultracentrifugation and ion-exchange chromatography. ROS-DNA showed single strand breaks, decrease in Tm, modification of thymine (58.3%) and guanine (20%). The mAb generated against ROS-DNA was characterized for antigen binding specificity by competition ELISA. Monoclonal antibody showed strong binding to ROS-modified DNA, its modified fragments, polynucleotides and bases. With the exception of native DNA, binding of unmodified polynucleotides and bases was much lower. The mAb distinctly recognized DNA samples from lymphocytes of healthy aged humans and gave maximum inhibitions of 49, 53, 64 and 70%, while not reacting with DNA from young population. Similarly, oxidative lesions in DNA from cancer patients were also efficiently recognized by the mAb. DNA from healthy controls served as negative control. The studies demonstrate that the mAb, although cross-reactive, preferentially binds ROS-modified epitopes on DNA. High reactivity of mAb to DNA samples from cancer patients and healthy aged humans indicates increased oxidative stress leading to DNA damage.

Aging↗
PubMed2000

Simultaneous detection and identification of O-GlcNAc-modified glycoproteins using liquid chromatography-tandem mass spectrometry.

Glycoproteins carrying O-linked N-acetylglucosamine (O-GlcNAc) modifications have been isolated from a wide range of organisms ranging from trypanosomes to humans. Interest in this modification is increasing as evidence accumulates that it is an abundant and transient modification that is dynamic and responsive to cellular stimuli. Concurrent advances in biological mass spectrometry (MS) have facilitated high-sensitivity protein identification by tandem MS. In this study, we show that the lability of the O-GlcNAc moiety to low-energy collision in tandem MS offers a means of distinguishing such peptides from others that are not modified. The differential between the energy required to remove the O-GlcNAc group and the energy required to fragment the peptide chain allows the O-GlcNAc group to be detected and the peptide sequence, and therefore the protein, to be identified. This technique thus allows the simultaneous detection and identification of O-GlcNAc-modified peptides, even when present at low levels in complex mixtures. The method was initially developed and validated using a synthetic O-GlcNAc-modified peptide and then applied to the detection of an extremely low abundance O-GlcNAc-modified peptide from bovine alpha-crystallin. We believe that with further development this assay system may prove to be a useful tool for the direct investigation of intracellular O-GlcNAc levels, thus providing valuable insights into the physiological role of O-GlcNAc modified proteins.

Acetylglucosamine↗
PubMed2004

Open tubular capillary electrochromatography of synthetic peptides on etched chemically modified columns.

Two sets of peptides, each having structurally similar amino acid sequences, have been investigated by capillary electrochromatography (CEC) using etched chemically modified capillaries as the separation medium. In comparison to gradient RP-HPLC, the resolving power of the described CEC methods has been found to be superior. A number of variables have been examined with respect to optimization of the separation of these closely related peptides with several different etched chemically modified capillaries. These experimental variables included the nature of the bonded moiety, the pH, the organic modifier type, and the amount of organic modifier in the buffer electrolyte. Systematic variation of these parameters results in significant changes in the migrational behavior of the investigated peptides and provides important insight into the underlying molecular separation processes that prevail in open tubular CEC. Moreover, under optimized conditions, efficient separations characterized by highly symmetrical peaks were achieved. In addition, this study has permitted the long-term stability as well as the short-term and long-term reproducibility of the etched chemically modified capillaries to be documented.

Amino Acid Sequence↗