Akt activation by arachidonic acid metabolism occurs via oxidation and inactivation of PTEN tumor suppressor.

Cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymes are overexpressed during inflammation and multistage tumor progression in many neoplastic disorders including lung, breast and pancreatic cancers. Here we report that the tumor suppressor phosphatase and tensin homolog (PTEN) is oxidized and inactivated during arachidonic acid (AA) metabolism in pancreatic cancer cell lines expressing COX-2 or 5-LOX. Oxidation of PTEN decreases its phosphatase activity, favoring increased phosphatidylinositol 3,4,5-triphosphate production, activation of Akt and phosphorylation of downstream Akt targets including GSK-3beta and S6K. These effects are recapitulated with pancreatic phospholipase A(2), which hydrolyses the release of membrane-bound AA. Interference with PTEN's physiological antagonism of signals from growth factors, insulin and oncogenes may confer risk for hypertrophic or neoplastic diseases associated with chronic inflammation or unwarranted oxidative metabolism of essential fatty acids.

Inhibition of gluconeogenesis by 2,5-anhydro-D-mannitol in isolated rat hepatocytes.

2,5-Anhydro-D-mannitol inhibited glucose synthesis, increased the pyruvate/phosphoenolpyruvate ratio and altered adenine nucleotide concentrations in hepatocytes isolated from fasted rats. The accumulations of 2,5-anhydro-D-mannitol 1,6-diphosphate, an allosteric activator of pyruvate kinase, and of ADP in treated hepatocytes can account for the increase in pyruvate/phosphoenolpyruvate ratio and the inhibition of glucose synthesis from lactate.

Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis.

We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer. The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis.

Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry.

We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.

Identification of phosphopeptides by mass spectrometry.

Phosphopeptides can be identified by ion spray mass spectrometry. The method was tested with phosphokemptide and with a proteolytic digest of one subunit (delta-subunit) of the nicotinic acetylcholine receptor. In the latter one peptide containing tyrosine phosphate, one with two serine phosphates, and two different peptides each containing one serine phosphate were unambiguously identified. Thus it is proven that ion spray mass spectrometry can be applied for the localization of phosphorylation sites in a known primary structure.

Collision cross sections for protein ions.

A method for the determination of cross sections for gas-phase protein ions, based on the energy loss of ions as they pass through a collision gas, is described. A simple model relates the energy loss to the number of collisions and hence the cross section. Results from a Monte Carlo model that support the validity of this approach are described. Experimental cross sections are reported for motilin, ubiquitin, cytochrome c, myoglobm, and bovine serum albumin. Cross sections range from approximately 800 Å(2) for motilin to approximately 14,000 Å(2) for bovine serum albumin and generally increase with the number of charges on the ion. Cytochrome c ions from aqueous solution show somewhat smaller cross sections than ions formed from solutions of higher organic content, suggesting that the gas-phase ions may retain some memory of their solution conformation.

Application of electrospray mass spectrometry in probing protein-protein and protein-ligand noncovalent interactions.

A novel mass spectrometry-based methodology using electrospray ionization (ESI) is described for the detection of protein-protein [interferon (IFN)-γ dimer] and protein-ligand [ras-guanosine diphosphate (GDP)] noncovalent interactions. The method utilizes ESI from aqueous solution at appropriate pH. The presence of the noncovalent complex of the IFN-γ dimer was confirmed by the observed average molecular weight of 33,819 Da. The key to the detection of the IFN-γ dimer is the use of an alkaline solution (pH ≈ 9) for sample preparation and for mass spectrornetry analysis. The effect of the declustering energy in the region of the ion sampling orifice and focusing quadrupole on the preservation of the gas-phase noncovalent complex (IFN-γ dimer) was also studied. The effect of the declustering energy on complex dissociation was further extended to probe the noncovalent protein-ligand association of ras-GDP. It was found that little energy is required to dissociate the IFN-γ dimer, whereas a substantial amount of energy is required to dissociate the gas-phase ras-GDP complex.

A trial of silver-zinc-allantoinate in the treatment of leg ulcers.

Three hundred thirty-nine of 400 chronic cutaneous ulcers in 264 patients (including some with multiple or bilateral ulcers of both) were healed with silver-zinc-allantoinate creamed (AZAC 1%). Some of the patients treated had failed to respond to medicated wrappings, saline dressings, and various other therapeutic agents, including antibiotics. In one week of treatment with AZAC 1%, bacterial counts were reduced on the average from approximately 2 X 10(7) to 2 X 10(5) (99%). Silver-zinc-allantoinate cream also demonstrated a wide spectrum of antimicrobial activity. It did not give rise to resistance by the infecting organisms, was nonallergenic, debrided necrotic tissue, and stimulated healthy granulation. Treatment was well tolerated, side effects being limited to a burning sensation in three patients. Most patients cared for themselves at home with minimal interference in their usual daily activities.

Low-dose heparin in postoperative patients: a prospective, coded study.

One hundred five patients over age 40 undergoing various major operations were randomly divided into control and treated groups; all were treated by subcutaneous injection containing either 5,000 international units aqueous heparin sodium or a placebo one hour prior to operation and every 12 hours thereafter for eight days. Deep vein thrombosis (DVT) was detected by daily -125I-fibrinogen injection and leg scanning, and confirmed by ascending phlebography. Both groups were comparably distributed by age, sex, variety of operation, incidence of previous thromboembolism, and myocardial and cerebrovascular disease. Blood loss was not increased in the treated group. Incidence of DVT was 8.6 percent for the total group, 7.5 percent in the heparin-tested group (four of 53), and 9.6 percent in the control group (five of 52), including one control patient with a normal scan (who later had a pulmonary embolus.

Distribution of zeranol in bovine tissues determined by selected ion monitoring capillary gas chromatography/mass spectrometry.

Bovine tissues, including liver, muscle, kidney, bile, serum, and urine, have been quantified by selected ion monitoring capillary gas chromatography/mass spectrometry to establish the distribution of the anabolic drug, zeranol, and its metabolites, taleranol and zearalanone, after administration of zeranol to 9 bovine animals. The method used to isolate, confirm, and quantify zeranol is undergoing validation by the United States Department of Agriculture, Food Safety Inspection Service (FSIS). Application of this method demonstrates utility in determining residue levels of zeranol in a variety of tissues with levels ranging over 4 orders of magnitude (i.e., 100 parts per trillion (ppt) to 1 part per million (ppm]. The analyte levels determined in this study complement previously reported pharmacokinetic data on the distribution of zeranol in addition to providing more specific information for taleranol and zearalanone. In this quantitative study it is shown that the liver is the main organ of deposition for zeranol, taleranol, and zearalanone, that taleranol is the main metabolite in the bovine, and that zeranol is efficiently eliminated.

Effects of duration of zilpaterol hydrochloride feeding and days on the finishing diet on feedlot cattle performance and carcass traits.

British and British x Continental steers (n = 560; initial BW = 339.4 +/- 1.76 kg) were used in a serial slaughter study with a completely random design to evaluate effects of zilpaterol hydrochloride (ZH; 8.33 mg/kg of dietary DM basis) on performance and carcass characteristics. Treatments were arranged in a 4 x 4 factorial (112 pens; 7 pens/treatment; 5 steers/pen) and included duration of ZH feeding (0, 20, 30, or 40 d before slaughter plus a 3-d ZH withdrawal period) and days on feed (DOF) before slaughter (136, 157, 177, and 198 d). No duration of ZH feeding x slaughter group interactions were detected for the performance measurements (P > 0.10). Final BW did not differ (P = 0.15) between the 0-d group and the average of the 3 ZH groups, but ADG was greater for the average of the 3 ZH groups during the period in which ZH diets were fed (P < 0.01) and for the overall feeding period (P = 0.05). As duration of ZH feeding increased, DMI decreased (P = 0.01) and G:F increased linearly (P < 0.01). With the exception of KPH (P = 0.022), no duration of ZH feeding x slaughter group interactions (P > 0.10) were detected for carcass characteristics. Regardless of the duration of ZH feeding, cattle fed ZH had greater HCW (P < 0.01), greater dressing percent (P < 0.01), less 12th-rib fat (P < 0.01), larger LM area (P < 0.01), less KPH (P = 0.03), and lower yield grade (P < 0.01) than the 0-d cattle. The 0-d group had greater marbling scores (P < 0.01) than cattle fed ZH diets, with a tendency for a linear decrease in marbling score (P = 0.10) as duration of ZH feeding was extended. A greater percentage of carcasses in the 0-d group graded USDA Choice or greater (P < 0.01) than in the 3 ZH groups, whereas the percentage of Select carcasses was greater (P = 0.01) for the 3 ZH groups. From d 0 to end (P = 0.04) and during the last 43 d on feed (P < 0.01), ADG responded quadratically to DOF before slaughter. No differences were detected among slaughter groups for DMI for the entire trial period; however, a quadratic response (P = 0.02) was observed for the final 43 d before slaughter. A quadratic response was also detected for the final 43 d before slaughter (P < 0.01) and from d 0 to end (P = 0.02) for G:F. Final BW, HCW, dressing percent, and 12th-rib fat increased linearly (P < 0.01) as DOF before slaughter increased. Our results indicate that no substantial effects on performance and carcass measurements were observed when ZH was fed for 30 or 40 d as opposed to 20 d, and that effects of ZH generally did not interact with DOF before slaughter.

Quantitative secondary ion monitoring gas chromatography/mass spectrometry of diethylstilbestrol in bovine liver.

A procedure is described for the extraction of diethylstilbestrol (DES) from animal tissue for quantitative capillary gas chromatography/mass spectrometry (GC/MS). The procedure is based upon use of a strong anion exchange polystyrene divinylbenzene resin for sample purification. The recovery of DES from the resin clean up was 88% in the high parts per trillion (ppt) range. Criteria for identification of DES using selected ion monitoring (SIM) GC/MS are presented. Liquid chromatography/mass spectrometry (LC/MS) was used to investigate altered DES cis/trans ratios observed in biological extracts.

Synthesis and testing of glucose derivatives for feeding to ruminants.

Glucose compounds were synthesized and tested for resistance in a 48-h in vitro rumen fermentation. Glucose complexed with formaldehyde, acetaldehyde, and acetone was resistant. When complexed with propionic and succinic acids, the products readily fermented. The resistant compounds also withstood acid hydrolysis at a pH similar to that of the abomasum. This suggests that these products would have little value as a dietary source of glucose for ruminants if the release of glucose to the animal depended on acid hydrolysis in the abomasum. The possibility of enzymatic hydrolysis of glucose-formaldehyde compound was studied in a feeding trial with sheep. Eight Finncross wethers were fed the formaldehyde product in three quantities in their diet. Blood and urine glucose plus blood, urine, and fecal total soluble carbohydrate were measured. It appears that this glucose-formaldehyde product resists rumen fermentation but does not become metabolically available to sheep as indicated by large quantities of product excreted in urine and feces.

Studies on the metabolism of omeprazole in the rat using liquid chromatography/ionspray mass spectrometry and the isotope cluster technique with [34S]omeprazole.

This paper describes the application of the ionspray (pneumatically-assisted electrospray) interface for liquid chromatography (LC) and atmospheric-pressure ionization mass spectrometry (API MS) to samples obtained in a study on the metabolism of omeprazole. In this study [34S]omeprazole was utilized for the stable isotope cluster technique. Over forty metabolites in a sample of partially purified rat urine were resolved by gradient elution LC with ionspray API MS detection, and each of them produced molecular ion 1:1 clusters (MH+ and [MH + 2]+). The chromatographic fidelity of the total-ion current (TIC) was excellent. The endogenous matrix of the sample was quite low, allowing a background-subtracted averaged mass spectrum of the entire TIC trace to produce a 'metabolite mass profile' depicting all the molecular ion 1:1 clusters in the sample. From this mass profile, it was possible to obtain direct information concerning oxygenation and conjugation reactions of the parent compound.