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.

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.

WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors. The physiological role of WSX-1, which is expressed mainly in T cells, was investigated in gene-targeted WSX-1-deficient mice. IFN-gamma production was reduced in isolated WSX-1(-/-) T cells subjected to primary stimulation in vitro to induce Th1 differentiation but was normal in fully differentiated and activated WSX-1(-/-) Th1 cells that had received secondary stimulation. WSX-1(-/-) mice were remarkably susceptible to Leishmania major infection, showing impaired IFN-gamma production early in the infection. However, IFN-gamma production during the later phases of the infection was not impaired in the knockout. WSX-1(-/-) mice also showed poorly differentiated granulomas with dispersed accumulations of mononuclear cells when infected with bacillus Calmette-Guerin (BCG). Thus, WSX-1 is essential for the initial mounting of Th1 responses but dispensable for their maintenance.

Application of a non-indexed dual sprayer pneumatically assisted electrospray source to the high throughput quantitation of target compounds in biological fluids.

The need for increased throughput in the quantitation of target compounds in biological fluids by high performance liquid chromatography/mass spectrometry continues to drive research in this area. This report describes the application of a prototype dual sprayer electrospray source for the quantitative analysis of biological samples. Quantitative performance for 180 compounds in a microsomal stability assay was found to be adequate when compared with a conventional single sprayer measurement. Issues with use of dual sprayers in a routine production environment are discussed.

Paragangliomas-- a decade of clinical experience.

BACKGROUND AND OBJECTIVES: This study is a review of 14 patients with paragangliomas between 1986 and 1996. The purpose was to determine the sites of origin, clinical manifestations and analyze the benefits of different treatment modalities. METHODS: There were 20 tumors in 14 patients. Three (21.0%) of the patients had familial history. There were 7 (50%) females and 7 (50%) males. Anatomically 14 (70%) tumors were in head and neck, 5 (25%) were in the retroperitoneum, one (5%) was in the heart. Of the head and neck tumors 9 (64.25%) were in the carotid body, 3 (21.42%) were found in the vagus, and 2(14.33%) were found in the middle ear. The tumor found in the heart was in the atrial septum. The clinical behavior of paragangliomas is determined by cellular characteristics, secreting capabilities and tumor location. The symptoms and signs depend on the site of origin and the stage at which it presents. The clinically functioning tumors were 3 (17%) in our experience and they typically present with uncontrolled hypertension. The carotid body and mediastinal tumors usually manifested as asymptomatic masses. The intravagal tumors presented with paresis of the nerve. Malignancy rarely occurs and is defined by the existence of metastasis rather than by histology. In our series 2 (10%) of the patients presented with metastasis to lymph nodes, and the vertebrae. The diagnoses in our patients were established by CT and MRI scanning. Angiography was performed in 5 patients with carotid body tumor, two of whom underwent therapeutic embolization to reduce the tumor size. The mainstay of treatment was surgical removal, though radiation has been advocated for patients who cannot undergo surgery. RESULTS: All patients underwent successful surgical resection of the tumor after appropriate preoperative preparation. Late mortality occurred in two (12.5%) patients at 3 and 5 years from unrelated etiology. Four (25%) patients were lost to follow-up. Three (18.7%) patients developed new primaries, two of them at two years and one after 8 years. One (6%) patient developed recurrent paraganglioma after remaining disease free for 20 years. CONCLUSION: In conclusion, paragangliomas are rare with multicentricity being more common in patients with familial history. The malignant potential of the tumor is determined by metastasis as there are no characteristic cellular change. Aggressive surgery is mandatory to obtain disease free survival with low morbidity and mortality. Recurrences can also be successfully operated with low morbidity.

Derivatization for electrospray ionization mass spectrometry. 3. Electrochemically ionizable derivatives.

In this paper, the use of ferrocene-based "electrochemically ionizable" derivatives to enhance ES-MS analysis of simple alcohols, sterols, and phenols is discussed. These derivatives are designed to take advantage of the electrolysis process inherent to operation of the ES ion source for selective ionization. Derivatization procedures, electrochemical character of the derivatives, and the ES-MS operational parameters necessary to maximize electrochemical ionization and to enhance gas-phase detection are presented with reference to ferrocenecarbamate ester derivatives of a variety of alcohol standards, as well as the ferroceneboronate derivative of the diol, pinacol. Tandem mass spectrometric analysis of the derivatives (precursor and product ion spectra) is shown to provide derivative confirmation, enhanced detection, and additional analyte structure information. The utility of this derivatization approach for the selective detection of alcohols in complicated mixtures is demonstrated using a saw palmetto (Serenoa repens) fruit extract known to contain a variety of alcohols at low levels.

Development and validation of sensitive method for determination of serum cotinine in smokers and nonsmokers by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry.

We describe a sensitive and specific method for measuring cotinine in serum by HPLC coupled to an atmospheric pressure chemical ionization tandem mass spectrometer. This method can analyze 100 samples/day on a routine basis, and its limit of detection of 50 ng/L makes it applicable to the analysis of samples from nonsmokers potentially exposed to environmental tobacco smoke. Analytical accuracy has been demonstrated from the analysis of NIST cotinine standards and from comparative analyses by both the current method and gas chromatography/high-resolution mass spectrometry. Precision has been examined through the repetitive analysis of a series of bench and blind QC materials. This method has been applied to the analysis of cotinine in serum samples collected as part of the Third National Health and Nutrition Examination Survey (NHANES III).

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.

Electrospray ionization mass spectrometry as a mechanistic tool: mass of human leucocyte elastase and a beta-lactam-derived E-I complex.

We have utilized liquid chromatography electrospray ionization mass spectrometry (ESI-MS) to probe the nature of the covalent E-I complex of human leucocyte elastase (HLE) and a beta-lactam. The mass spectrum of HLE isozyme 4 displayed one major and two minor components with masses of 25,202, 25,043, and 24,522 Da, respectively. Isozyme 3 displayed three components, with masses of 25,180, 24,030, and 24,523 Da. These data suggest that the isozymes differ in the type and not the content of carbohydrate. The minor components represent decreases in carbohydrate content. Inactivation of isozyme 4 with trans-4-(ethoxycarbonyl)-3-ethyl-1-[(4-nitrophenyl)sulfonyl]-azetidin -3-one increased the mass of the three components by that of the parent compound. Similar results were obtained with the mixture of HLE isozymes. These observations demonstrate that HLE does not catalyze the beta-elimination of p-nitrophenylsulfinate as Firestone et al. [(1990) Tetrahedron 46, 2255) suggested. In addition, it suggests that a "double hit" of both the active-site serine and histidine is not required to form a stable acyl-enzyme. Noncovalent complexes between HLE and either the tight-binding secretory leucoprotease inhibitor (SLPI) or a slow tight-binding peptide difluoroketone inhibitor were not observed by ESI-MS. SLPI displayed a mass of 11,710 Da in the absence and presence of HLE. These data demonstrate the utility of ESI-MS to probe the mechanism of inhibition of enzymes by mechanism-based inhibitors.

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.

Identification of the steroids in neonatal plasma that interfere with 17 alpha-hydroxyprogesterone radioimmunoassays.

Neonatal plasma contains interferents that increase the apparent 17 alpha-hydroxyprogesterone (17-OHP) content measured by direct (no-extraction) radioimmunoassay. We fractionated extracts from neonatal plasma pools by liquid chromatography with a Sephadex LH-20 column and measured 17-OHP immunoreactivity by a direct test kit. We found immunoreactivity in the free steroid and glucuronide fraction and also in the monosulfate fraction. We analyzed these two fractions by reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry. We collected fractions and assayed for 17-OHP immunoreactivity. The HPLC fractions containing the interfering steroid monosulfates were analyzed by ion-spray mass spectrometry and, after solvolysis, by gas chromatography-mass spectrometry. Several monosulfates were identified, including those of 17 alpha-hydroxy-pregnenolone, 16 alpha-hydroxypregnenolone, pregnenolone, and several pregnenetriols. 17 alpha-Hydroxypregnenolone sulfate was the most significant interferent. Other commercially available 17-OHP assays showed similar interference when used without an extraction step. Kit manufacturers should select antibodies and protocols to minimize cross-reaction with sulfates, especially 17 alpha-hydroxypregnenolone sulfate.

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.

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.

Determination of L-365,260, a new cholecystokinin receptor (CCK-B) antagonist, in plasma by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry.

L-365,260 is a novel cholecystokinin receptor antagonist. A sensitive and specific liquid chromatographic/mass spectrometric assay has been developed for the determination of the drug in plasma using the CD3-labeled species as the internal standard. Plasma extracts were separated on a 3 cm C18 reverse-phase high-performance liquid chromatography column. The column eluate passed, by means of a heated nebulizer interface, into a corona discharge atmospheric chemical ionization source. The mass spectrometer was operated in the positive ion tandem mass spectrometric mode. The method has sufficient sensitivity, specificity, precision, accuracy and selectivity for the determination of drug concentrations in clinical samples. The chromatographic run time is less than 2 min.

Characterization of the tryptic map of recombinant DNA derived tissue plasminogen activator by high-performance liquid chromatography-electrospray ionization mass spectrometry.

A detailed tryptic map is presented for recombinant human tissue plasminogen activator (rt-PA). Electrospray ionization mass spectrometry is utilized as an on-line HPLC detector for tryptic mapping of this glycoprotein. The additional dimension provided by mass spectrometry gives considerably more detail about the complex tryptic map and significantly enhances the high-resolution chromatographic separation by distinguishing by mass any coeluting components. Through this improvement, the proline isomers of a tryptic peptide were observed eluting over a broad range of retention times. The glycopeptides of rt-PA are observed as well as any corresponding nonglycosylated peptides. In addition, the carbohydrate heterogeneity is readily observed, allowing analysis of the carbohydrate composition. The characteristic diagonal patterns formed by glycopeptides in a contour plot of the data allow rapid recognition of the glycopeptides.

Structural characterization of protein tryptic peptides via liquid chromatography/mass spectrometry and collision-induced dissociation of their doubly charged molecular ions.

The formation of multiply charged molecular ions via the field-assisted ion evaporation mechanism during electrospray ionization enables the use of an atmospheric pressure ionization quadrupole mass spectrometer system for characterizing biologically important peptides. The straightforward implementation of high-performance liquid chromatography (HPLC) into this new strategy to determine the molecular weight of tryptic peptides via the pneumatically assisted electrospray (ion spray) interface is presented. Examples utilizing both microbore (1.0 mm) and standard bore (4.6 mm) inside diameter columns are shown for the LC/MS molecular weight determination of tryptic peptides in methionyl-human growth hormone (met-hGH). Injected levels from 50 to 75 pmol of tryptic digest onto 1 mm i.d. HPLC columns provided full-scan LC/MS or LC/MS/MS results without postcolumn splitting of the effluent. When standard 4.6 mm i.d. HPLC columns were used, a 20:1 postcolumn split was utilized, which required from 1 to 5 nmol of injected tryptic digest for full-scan LC/MS or LC/MS/MS results. Collision-induced dissociation (CID) mass spectra resulting from either "infusion" or on-line LC/MS/MS analysis of the abundant doubly charged ions that predominate for tryptic peptides under electrospray conditions provided structurally useful sequence information for met-hGH and human hemoglobin tryptic digests. The slower mass spectrometer scan rate used during infusion of sample provides more accurate mass assignments than on-line LC/MS or LC/MS/MS, but the latter on-line experiments preclude ambiguities caused by matrix or component interferences. However, in some instances very weak CID product ions preclude complete tryptic peptide structural characterization based upon the CID data alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of proteins and glycoproteins at the picomole level by on-line coupling of microbore high-performance liquid chromatography with flow fast atom bombardment and electrospray mass spectrometry: a comparative evaluation.

A comparison of continuous-flow fast atom bombardment (FAB) mass spectrometry and nebulization-assisted electrospray for analysis of proteins and glycoproteins by high-performance liquid chromatography (HPLC)/mass spectrometry is presented. The evaluation was made using enzymatic digests of recombinant soluble CD4 glycoprotein and recombinant hepatitis B surface antigen and a mixture of HPLC retention standard peptides. These samples were analyzed by reversed-phase HPLC on a microbore (1 x 100 mm C18) gradient HPLC system with 10% or 20% of the eluent containing approximately 20-100 pmol of the sample directed into the continuous-flow FAB mass spectrometric or electrospray mass spectrometric source, respectively. The techniques were evaluated on the criteria of chromatographic integrity, ease of data analysis, protein sequence coverage, discrimination effects, ability to detect glycopeptides, simplicity of operation, and sensitivity. Both techniques produce useful peptide molecular weight data from comparable amounts of sample injected. However, the nebulization-assisted electrospray system is capable of yielding higher peptide mapping coverages with the least sample consumed in toto due in part to the wider mass ranges resulting from the multicharging effect and to the ability to detect glycopeptides. Under the experimental conditions employed here no fragmentation was observed in the electrospray mass spectra. In contrast, significant, sequence informative fragmentation was occasionally observed for peptides in the continuous-flow FAB mass spectral data.