Cholesterol biosynthesis regulation and protein changes in rat liver following treatment with fluvastatin.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulator in cholesterol biosynthesis and HMG CoA reductase inhibitors (statins) have become a widely prescribed family of lipid lowering agents. Cholesterol synthesis occurs predominantly in liver which is the target organ of statins. We studied the effects of fluvastatin (Lescol), a member of the statin family, on hepatic protein regulation. Male F344 rats treated with 0.8 mg/kg per day fluvastatin or 24 mg/kg per day fluvastatin for 7 days showed treatment-related changes in 58 liver proteins (P<0.005). Major effects were evident in the cholesterol biosynthesis pathway including the induction of enzymes upstream and downstream of the target enzyme HMG CoA reductase. Treatment also triggered alterations in key enzymes of carbohydrate metabolism and was associated with changes in a heterogeneous set of cellular stress proteins involved in cytoskeletal structure, calcium homeostasis and protease activity. The latter set of protein alterations indicates that hepatotoxicity is associated with high-dose treatment. Based on the results it is suggested that HMG-CoA synthase and isopentenyl-diphosphate delta-isomerase may be explored as alternative drug targets and that the induction levels of these enzymes may serve as a measure of potency of individual statin drugs. It is proposed that efficacy and cellular stress markers discovered in this study may be used in a high throughput screen (HTS) assay format to compare efficiently and accurately the therapeutic windows of different members of the statin family.

Sequence information, distinction and quantitation of C-terminal leucine and isoleucine in ternary complexes of tripeptides with Cu(II) and 2,2'-bipyridine.

Tripeptides form ternary complexes with Cu(2+) and 2,2'-bipyridine (bpy) that self-assemble upon mixing the components in aqueous methanol solution. Electrospray ionization (ESI) of the complex solutions provides abundant singly charged [Cu(peptide -- H)bpy](+) and doubly charged [Cu(peptide)bpy](2+) ions. Collision-induced dissociation (CID) at low ion kinetic energies of several tripeptides, AGG, GGA, LGG, GGL, GGI, FGG, GGF, LGF, GLF, GFL, GYA and GAY, showed fragments that were indicative of the amino acid sequence in the peptide. In addition, CID of single and doubly charged complexes of isomeric tripeptides GGL and GGI provided unambiguous distinction of the isomeric leucine and isoleucine residues. Leucine peptides eliminated C(3)H(7) radicals from the amino acid side-chain whereas isoleucine eliminated C(2)H(5) radicals. CID of gas-phase doubly charged peptide complexes in a quadrupole ion trap produced a series of singly charged sequence fragments that following isolation and further CID furnished distinct fragments that allowed quantitation of leucine and isoleucine-containing peptides in mixtures.

Proteomics to display lovastatin-induced protein and pathway regulation in rat liver.

Lovastatin is a lipid lowering agent that acts by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a key regulatory enzyme in cholesterol biosynthesis. In this study the pattern of gene network regulation induced in hepatic proteins as a response to lovastatin treatment was analyzed by proteomics. In livers of male F344 rats treated with 1.6 mg/kg/day lovastatin or 150 mg/kg/day lovastatin for seven days, 36 proteins were found to be significantly altered (p<0.001) in relation to treatment. The changed proteins were classified according to their cellular function and participation in biochemical pathways. The following observations were made: (i) inhibition of HMG-CoA reductase provoked a regulatory response in the cholesterol synthesis pathway including the induction of cytosolic HMG-CoA synthase and of isopentenyl-diphosphate delta-isomerase, (ii) manipulation of the lipid metabolism triggered alterations in key enzymes of the carbohydrate metabolism, and (iii) lovastatin treatment was associated with signs of toxicity as reflected by changes in a heterogeneous set of cellular stress proteins involved in functions such as cytoskeletal structure, calcium homeostasis, protease inhibition, cell signaling or apoptosis. These results present new insights into liver gene network regulations induced by lovastatin and illustrate a yet unexplored application of proteomics to discover new targets by analysis of existing drugs and the pathways that they regulate.

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry.

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.

Quantitative electrospray ionization mass spectrometric studies of ternary complexes of amino acids with Cu(2+) and phenanthroline.

Electrospray ionization (ESI) mass spectra of ternary complexes of Cu(2+) and 1,10-phenanthroline with the 20 essential amino acids (AA) were investigated quantitatively. Non-basic amino acids formed singly charged complexes of the [Cu(AA - H)phen](+) type. Lysine (Lys) and arginine (Arg) formed doubly charged complexes of the [Cu(HAA - H)phen](2+) type. Detection limits were determined for the complexes of phenylalanine (Phe), glutamic acid (Glu) and Arg, which were at low micromolar or submicromolar concentrations under routine conditions. Detection limits of low nanomolar concentrations are possible for amino acids with hydrophobic side-chains (Phe, Tyr, Trp, Leu, Ile) as determined for Phe. The efficiencies for the formation by ESI of gaseous [Cu(AA - H)phen](+) ions were determined and correlated with the acid-base properties of the amino acids, ternary complex stability constants and amino acid hydrophobicities expressed as the Bull-Breese indices (DeltaF). A weak correlation was found between DeltaF and the ESI efficiencies for the formation of gaseous [Cu(AA - H)phen](+) [Cu(HAA - H]phen](2+) and [AA + H](+) ions that showed that amino acids with hydrophobic side-chains were ionized more efficiently. In the ESI of binary and ternary amino acid mixtures, the formation of gas-phase Cu-phen complexes of amino acids with hydrophobic side-chains was enhanced in the presence of complexes of amino acids with polar or basic side-chains. An interesting enhancement of the ESI formation of [Cu(Glu - H)phen](+) was observed in mixtures. The effect is explained by ion-cluster formation at the droplet interface that results in enhanced desorption of the glutamic acid complex.

TGF-beta enhances osteoclast differentiation in hematopoietic cell cultures stimulated with RANKL and M-CSF.

TGF-beta has been shown to inhibit and stimulate osteoclastogenesis. The purpose of this study was to evaluate the effects of TGF-beta in hematopoietic cell cultures stimulated with RANKL and M-CSF. In cocultures of hematopoietic cells and BALC cells (a calvarial-derived cell line), TGF-beta inhibited tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell formation. In contrast, TGF-beta enhanced TRAP-positive multinucleated cell formation up to 10-fold in hematopoietic cell cultures containing few osteoblastic/stromal cells. Likewise, TGF-beta increased the number of calcitonin receptor (CTR)-positive multinucleated and mononucleated cells in a concentration-dependent manner. An increase in cell size and multinuclearity was also observed in the presence of TGF-beta. The stimulatory effects of TGF-beta were dependent on the presence of M-CSF and RANKL. When differentiated on bovine cortical bone slices, these cells formed resorption lacunae. These results suggest that TGF-beta has a direct stimulatory effect on osteoclastogenesis in hematopoietic cells treated with RANKL and M-CSF.

Mass spectrometric analysis of catechol-histidine adducts from insect cuticle.

Adducts of catechols and histidine, which are produced by reactions of 1,2-quinones and p-quinone methides with histidyl residues in proteins incorporated into the insect exoskeleton, were characterized using electrospray ionization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ESMS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITMS). Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtained from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H]+ ion at m/z 308, rather than the expected m/z 307. High-resolution fast atom bombardment MS yielded an empirical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3, 4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] instead of a DA adduct. Similar results were obtained when histidyl-catechol compounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOPET derivative. Direct MS analysis of unfractionated cuticle hydrolysate revealed intense parent and product ions characteristic of 6- and 7-linked adducts of histidine and DOPET. Mass spectrometric analysis of model adducts synthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H]+ of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') isomer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cuticular proteins, and both ring and side-chain carbons of three catechols: NADA, N-beta-alanyldopamine, and DOPET.

Computed axial tomography of the porcine nasal cavity and a morphometric comparison of the nasal turbinates with other visualization techniques.

A non-invasive imaging modality, computed tomography (CT), was used to visualize changes in nasal turbinates of anesthetized pigs over a 12-week observation period (pigs were 14 wk of age at study week 0). Normal, non-infected pigs were compared to pigs with mild challenge-induced atrophic rhinitis (AR) in order to detect subtle differences in morphology. To determine feasibility for time course studies in future experiments, morphometric quantitation at the level of the 2nd premolar (turbinate area ratio or TAR) in cross-section CT images at multiple timepoints was done. Additionally, at study termination, the TAR determined from CT images, magnetic resonance imaging (MRI), and wet tissue (WT), were compared to each other and to the standard subjective measure, visual scoring. There were no statistically significant differences between the control and AR groups at CT imaging dates of 0, 3, 6, 9, or 12 wk (P = 0.182). However, a statistically significant decrease in TAR measurements over time (P = 0.015) was observed in both groups, with lower mean values observed on Weeks 3 and 6 before rebounding to baseline values at study termination. At Week 12 (termination of the study), the TAR measurements derived from CT, MRI, and WT were not statistically different from one another (P = 0.220) and the treatment group-by-method interaction was not significant (P = 0.800). This provided evidence of equivalency of the techniques. Mean values for normal and infected groups were not significantly different based on either TAR imaging methods (P = 0.552) or visual scores (P = 0.088). Thus, the current study demonstrated that CT was an acceptable alternative imaging modality which could be used for quantitation of turbinate changes in snouts of live pigs to provide data comparable to tissue taken at necropsy. Computed tomographic imaging would allow non-invasive tracking of disease or treatment responses within individual animals over time. Morphometric analysis of the TAR was equivalent between the CT, MRI, and WT specimens.

Method to compare collision-induced dissociation spectra of peptides: potential for library searching and subtractive analysis.

We report the development of a method to compare collision-induced dissociation (CID) spectra of peptides. This method employs a cross-correlation analysis of a CID spectrum to a reference spectrum and normalizes the cross-correlation score to the autocorrelation of the CID spectra. The query spectrum is compared by using both mass information and fragmentation patterns. Fragmentation patterns are compared to each other using a correlation function. To evaluate the specificity of the approach, a set of 2180 tandem mass spectra obtained from both triple-quadrupole tandem mass spectrometers (TSQ) and quadrupole ion trap mass spectrometers (LCQ) was created. Comparisons are performed between tandem mass spectra obtained on the same instrument type as well as between different instrument types. Accurate and reliable comparisons are demonstrated in both types of analyses. The scores obtained in the cross-comparison of TSQ and LCQ tandem mass spectra of the same peptide are found to be slightly lower than comparisons performed with spectra obtained on the same instrument type. The method appears insensitive to variations in day-to-day performance of the instrument, minor variations in fragment ion abundance, and instrumental differences inherent in the same instrument model. The use of this method of comparison is demonstrated for library searching and subtractive analysis of tandem mass spectra obtained during LC/MS/MS experiments.

Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry.

Conventional capillary liquid chromatography/mass spectrometry (LC/MS) typically employs low microl/min flow rates with gas/liquid sheath to enhance spray stability. Over the past several years a number of reports have demonstrated success with electrospray (ES) interface designs optimized for submicroliter/min flows which have clear advantages in terms of enhancement of detection limit, lower sample consumption, and ability to accommodate a wider range of buffer conditions. We report here a fritless electrospray interface (FESI) design that is inexpensive and robust and can be operated and adapted to accommodate a variety of applications for submicroliter/min flow rates. The novelty of this interface revolves around the use of a fritless microcapillary column and precolumn application of electrospray voltage at a microtee junction to achieve stable microspray and nanospray flow rates. This sheathless FESI device eliminates postcolumn dead volume since small particles (

Quantitation of glandular gastric changes in rats given a proton pump inhibitor for 3 months with emphasis on sampling scheme selection.

Proton pump inhibitors and H2-receptor antagonists suppress gastric acid secretion and secondarily induce hypergastrinemia. Sustained hypergastrinemia has a trophic effect on stomach fundic mucosa, including enterochromaffin-like (ECL) cell hypertrophy and hyperplasia. Histomorphometric quantitation of the pharmacologic gastric effects was conducted on 10 male and 10 female rats treated orally with LY307640 sodium, a proton pump inhibitor, at daily doses of 25, 60, 130, or 300 mg/kg for 3 mo. Histologic sections of glandular stomach, stained for chromogranin A, were evaluated by image analysis to determine stomach mucosal thickness, mucosal and nonmucosal (submucosa and muscularis) area, gastric glandular area, ECL cell number/area and cross-sectional area. Total mucosal and nonmucosal tissue volumes per animal were derived from glandular stomach volumetric and area data. Daily oral doses of compound LY307640 sodium caused slight to moderate dose-related mucosal hypertrophy and ECL cell hypertrophy and hyperplasia in all treatment groups as compared with controls. All observed effects were prominent in both sexes but were generally greater in females. The morphometric sampling schemes were explored to optimize the data collection efficiency for future studies. A comparison between the sampling schemes used in this study and alternative schemes was conducted by estimating the probability of detecting a specific percentage of change between the male control and high-dose groups based on Tukey's trend test. The sampling scheme analysis indicated that mucosal thickness and mass had been oversampled. ECL cell density quantitation efficiency would have been increased by sampling the basal mucosa only for short-term studies. The ECL cell size sampling scheme was deemed appropriate for this type of study.

The quantitation of turbinate atrophy in pigs to measure the severity of induced atrophic rhinitis.

The two-fold purpose of this study was to establish a useful image analysis technique for quantitation of turbinate atrophy and to determine an optimum bacterial dose for inducing atrophic rhinitis (AR). Two morphometric analysis methods were compared to determine a turbinate area ratio (TAR) and a turbinate perimeter ratio (TPR); the ratios of turbinate area to total nostril area and of turbinate perimeter to total nostril perimeter, respectively. Our first image analysis method differed from Collins et al (1) in that we used direct image capture (digitalization) via a video camera and a Macintosh microcomputer, rather than photographs and a digitizer tablet. The tracing techniques were the same as those used by Collins et al. The second morphometric method was modified from the first by exclusion of dorsal turbinate when tracing the nostril area and directly tracing only the ventral turbinate to get a turbinate measurement without subtracting. Area and perimeter ratios, for both methods, were compared to conventional visual snout scores, ventral measurements, and to each other. The results of the two image analysis methods correlated well, both with each other and with the visual scores. Doses of Pasteurella multocida (Pm) at a constant level, and Bordetella bronchiseptica (Bb) at various concentrations, were administered to 36 Hampshire-Duroc F1 SPF pigs to determine the best dose and frequency for inducing AR. Although the dose selection may have been somewhat affected by the pre-existing presence of Bb, the optimal dose per naris in this study was 2 mL Bb at 10(7) cfu/mL combined with 2 mL Pm at 10(9) cfu/mL inoculum. The frequency of administration (1 x or 2 x) did not greatly affect results. Turbinate area ratio was the best tool for quantitating gross morphological turbinate changes associated with atrophic rhinitis in this study. Our simplified modification of Collins et al image analysis method (exclusion of dorsal turbinates and direct measurement of ventral turbinates) correlated well with visual scores, and, when compared to Collins et al method, required less data manipulation and labour.

Carboxylate and amine terminus directed fragmentations in gaseous dipeptide complexes with copper (II) and diimine ligands formed by electrospray.

Singly and doubly charged peptide complexes with copper(II) and 2,2'-bipyridyl (bpy) are formed in the gas phase by electrospraying water-methanol solutions containing the components. Collisionally activated dissociations at low kinetic energies of singly charged complexes of the [CuII(peptide-H)(bpy)].+ type provide information about the amino acid sequence for L-Phe-Leu, L-Leu-Phe, L-Phe-Pro, L-Pro-Phe, L-Phe-Met, L-Met-Phe, L-Ser-Phe, L-Asp-Phe, and L-His-Phe. Dissociations of doubly charged complexes of the [CuII(peptide)(bpy).2+ type also allow identification of the N- and C-terminal amino acid residues. Leucine and isoleucine residues are readily distinguished in L-Ala-Leu and L-Ala-Ile through dissociations of their Cu complexes. Ion dissociation mechanisms, as elucidated by deuterium labeling, are discussed.

Point counting on the Macintosh. A semiautomated image analysis technique.

In image analysis, point counting is used to estimate three-dimensional quantitative parameters from sets of measurements made on two-dimensional images. Point counting is normally conducted either by hand only or manually through a planimeter. We developed a semiautomated, Macintosh-based method of point counting. This technique could be useful for any point counting application in which the image can be digitized. We utilized this technique to demonstrate increased vacuolation in white matter tracts of rat brains, but it could be used on many other types of tissue. Volume fractions of vacuoles within the corpus callosum of rat brains were determined by analyzing images of histologic sections. A stereologic grid was constructed using the Claris MacDraw II software. The grid was modified for optimum line density and size in Adobe Photoshop, electronically superimposed onto the images and sampled using version 1.37 of NIH Image public domain software. This technique was further automated by the creation of a macro (small program) to create the grid, overlay the grid on a predetermined image, threshold the objects of interest and count thresholded objects at intersections of the grid lines. This method is expected to significantly reduce the amount of time required to conduct point counting and to improve the consistency of counts.