Modulation of behavioral networks by selective interneuronal inactivation.

Gamma-aminobutyric acid (GABA)-ergic disturbances are hallmark features of schizophrenia and other neuropsychiatric disorders and encompass multiple interneuronal cell types. Using bacterial artificial chromosome-driven, miRNA silencing technology we generated transgenic mouse lines that suppress glutamic acid decarboxylase 1 (GAD1) in either cholecystokinin (CCK)- or neuropeptide Y (NPY)-expressing interneurons. In situ lipidomic and proteomic analyses on brain tissue sections revealed distinct, brain region-specific profiles in each transgenic line. Behavioral analyses revealed that suppression of GAD1 in CCK+ interneurons resulted in locomotor and olfactory sensory changes, whereas suppression in NPY+ interneurons affected anxiety-related behaviors and social interaction. Both transgenic mouse lines had altered sensitivity to amphetamine albeit in opposite directions. Together, these data argue that reduced GAD1 expression leads to altered molecular and behavioral profiles in a cell type-dependent manner, and that these subpopulations of interneurons are strong and opposing modulators of dopamine system function. Furthermore, our findings also support the hypothesis that neuronal networks are differentially controlled by diverse inhibitory subnetworks.

MALDI MS imaging as a powerful tool for investigating synovial tissue.

OBJECTIVE: To identify and image protein biomarker candidates in the synovial tissue of patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA). METHODS: A novel matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) technique was applied to the analysis of synovial tissue. Patients were classified according to the American College of Rheumatology (ACR) criteria for RA. Frozen sections were stained to obtain morphological data. Serial sections were desiccated, and spotted with matrix for MALDI analysis. Ions generated by laser irradiation of the tissue were separated in time, based on their m/z ratio, and were subsequently detected. IMS was used in a 'profiling' mode to detect discrete spots for rapid evaluation of proteomic patterns in various tissue compartments. Photomicrographs of the stained tissue images were reviewed by a pathologist. Areas of interest (10 discrete areas/compartment) were marked digitally and the histology-annotated images were merged to form a photomicrograph of the section taken before the MALDI measurement. Pixel coordinates of these areas were transferred to a robotic spotter, the matrix was spotted, and the coordinates of the spots were transferred to a mass spectrometer for spectral acquisition. The data generated were then subjected to biocomputation analysis to reveal the biomarker candidates. RESULTS: Several peaks (m/z) consistent in mass with calgranulins, defensins, and thymosins were detected and their distribution in various synovial compartments (synovial lining and sublining layer) was demonstrated. CONCLUSION: MALDI IMS is a powerful tool for the rapid detection of numerous proteins (in situ proteomics) and was applied here for the analysis of the distribution of proteins in synovial tissue sections.

Optimization of a MALDI-Imaging protocol for studying adipose tissue-associated disorders.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is increasingly recognized for its potential in the discovery of novel biomarkers directly from tissue sections. However, there are no MALDI IMS studies as yet on the adipose tissue, a lipid-enriched tissue that plays a pivotal role in the development of obesity-associated disorders. Herein, we aimed at developing an optimized method for analyzing adipose tissue lipid composition under both physiological and pathological conditions by MALDI IMS. Our studies showed an exacerbated lipid delocalization from adipose tissue sections when conventional strategies were applied. However, our optimized method using conductive-tape sampling and 2,5-dihydroxybenzoic acid (DHB) as a matrix, preserved the anatomical organization and minimized lipid diffusion from sample sections. This method enabled the identification of a total of 625 down-regulated and 328 up-regulated m/z values in the adipose tissue from a rat model of extreme obesity as compared to lean animals. Combination of MALDI IMS and liquid chromatography (LC)-MS/MS data identified 44 differentially expressed lipid species between lean and obese animals, including phospholipids and sphingomyelins. Among the lipids identified, SM(d18:0_18:2), PE(P-16:0_20:0), and PC(O-16:0_16:1) showed a differential spatial distribution in the adipose tissue of lean vs. obese animals. In sum, our method provides a valuable new tool for research on adipose tissue that may pave the way for the identification of novel biomarkers of obesity and metabolic disease.

A sperm cytoskeletal protein that signals oocyte meiotic maturation and ovulation.

Caenorhabditis elegans oocytes, like those of most animals, arrest during meiotic prophase. Sperm promote the resumption of meiosis (maturation) and contraction of smooth muscle-like gonadal sheath cells, which are required for ovulation. We show that the major sperm cytoskeletal protein (MSP) is a bipartite signal for oocyte maturation and sheath contraction. MSP also functions in sperm locomotion, playing a role analogous to actin. Thus, during evolution, MSP has acquired extracellular signaling and intracellular cytoskeletal functions for reproduction. Proteins with MSP-like domains are found in plants, fungi, and other animals, suggesting that related signaling functions may exist in other phyla.

Human urinary metabolites of 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftoraful).

Two hydroxylated metabolites were isolated from the urine of a patient who had received ftorafur (5 g/sq m). These metabolites were identified by mass spectrometry and nuclear magnetic resonance spectroscopy as trans-3'- and cis-4'-hydroxyftorafur. The compounds were not converted to 4-fluorouracil when incubated in plasma, base (pH 9), or water. Because of their stability, it is unlikely that these metabolites are in vivo precursors of 5-fluorouracil. There are indications that less stable, unisolatable, hydroxylated ftorafur derivatives are intermediates in the conversion of ftorafur to 5-fluorouracil.

Phagocytosis, cellular distribution, and carcinogenic activity of particulate nickel compounds in tissue culture.

The uptake, toxicity, and morphological transformation efficacy of various water-insoluble nickel compounds were examined in tissue culture. Particles (2.2 to 4.8 micrometers) of crystalline Ni3S2, crystalline NiS, and crystalline Ni3Se2 were actively phagocytized by cultured cells as determined by light and electron microscopy. However, particles of similar size consisting of amorphous NiS and metallic nickel were not significantly phagocytized despite long exposure periods to high concentrations. X-ray fluorescence spectrometry measurements of metal levels in subcellular fractions isolated from cells treated with crystalline Ni3S2, crystalline NiS, or amorphous NiS confirmed that amorphous NiS did not significantly enter the cells, either as a phagocytized particle or in a solubilized form, while the other two crystalline nickel compounds were actively taken up. Cells treated with amorphous NiS contained nickel levels generally less than 10% of the nickel levels in whole cells and in cytoplasmic fractions, or nuclear fractions of cells treated with either crystalline NiS or crystalline Ni3S2. The phagocytized nickel particles were always observed in the cytoplasm with light and electron microscopy, but substantial nickel levels were measured in the nuclear fraction. These and other results suggest that the nickel particles were broken down in the cytoplasm to a size range no longer detectable with the electron microscope and then subsequently entered the nucleus. Control experiments suggest that at least 20% of the nickel measured in the nucleus isolated from cells treated with Ni3S2 is no longer part of a sedimentable particle with the same particle size and/or solubility properties of the parent compound. A substantial portion of the nickel associated with the nuclear fraction coprecipitated with trichloroacetic acid-insoluble material, suggesting that nickel binds strongly to cellular macromolecules. The phagocytized particulate nickel compounds were more cytotoxic as determined by reduction of cell-plating efficiency and induced more morphological transformations in the Syrian hamster embryo cell transformation assay than did the particulate nickel compounds which were not phagocytized. Manganese dust inhibited the morphological transformation induced by Ni3S2 and also reduced the phagocytosis of Ni3S2 particles.

Clinical pharmacology of intraarterial cis-diamminedichloroplatinum(II).

After intraarterial (30 patients) or i.v. (seven patients) administration of cis-diamminedichloroplatinum, X-ray fluorescence spectrometry was used to measure platinum concentrations in plasma and urine. Arteries infused included hepatic (seven patients), carotid (six patients), iliac (ten patients), brachial (three patients), and femoral (four patients). All patients received i.v. mannitol. Pharmacokinetic parameters after intraarterial administration were similar to those after i.v. administration, although differences existed for different intraarterial routes of administration. Mean for all patients combined were: Co, 2.67 +/- 0.97 (S.D.) microgram/ml; t1/2 beta, 71.1 +/- 26.6 hr; clearance, 0.72 +/- 0.25 liters/hr/sq m; total volume of distribution, 45.2 +/- 17.0 liters/sq m; C x t, 167 +/- 72 mg/hr/liter; and 24-hr urinary excretion, 20 +/- 10% of the administered dose. Intrahepatic infusion of the drug was associated with a significantly lower Co (1.88 +/- 0.50 g/ml) and C x t (140 +/- 25 mg hr/liter) and significantly higher clearance (0.91 +/- 0.24 liters/hr/sq m) and volume of distribution (67.6 +/- 4.6 liters/sq m) than administration by other routes, suggesting first pass extraction of drug by liver. In addition, an apparent minor late rise in serum platinum concentration may suggest enterohepatic recirculation of drug. High fluid intake was associated with a low Co and a high volume of distribution, consistent with expansion of the central compartment by fluids. Low serum albumin (less than 3.5 g/dl) was associated with significant shortening of the t1/2 beta (50.5 +/- 21.6 hr), suggesting that the amount of unbound filterable drug may possibly be higher in patients with low serum albumin concentrations. Plasma from veins draining an infused area has a higher Co and C x t during infusion than concurrent plasma from peripheral veins. Thus, intraarterial administration of cis-diamminedichloroplatinum results in increased drug exposure of tumor in the infused area without substantially decreasing exposure of systemic tumor.

A probasin-large T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential.

Neuroendocrine (NE) cells may be involved not only in growth and differentiation of the normal prostate but also in carcinogenesis and progression of prostate adenocarcinoma (Pca), including development of androgen resistance. However, the exact pathophysiology of NE cells in Pca remains poorly understood. Here we describe a transgenic model of Pca with progressive NE differentiation. Seven lines of transgenic mice with the rat prostate-specific large probasin promoter linked to the SV40-large T antigen (Tag) that develop prostatic neoplasia have been established. In this study, one of the seven lines (12T-10) was characterized by examination of 52 mice aged from 2-12 months. With advancing age, low-grade prostatic intraepithelial neoplasia, high-grade prostatic intraepithelial neoplasia, microinvasion, invasive carcinoma, and poorly or undifferentiated carcinoma with NE differentiation appeared in the prostates in sequential order. Whereas Tag is expressed uniformly in prostate epithelium, only an increasing subset of cells in prostatic intraepithelial neoplasia showed NE differentiation by chromogranin immunostaining. Frankly invasive carcinoma developing subsequently showed occasional definitive glandular differentiation (adenocarcinoma) and particularly undifferentiated carcinoma with NE histological features similar to those observed in NE carcinomas in humans. The NE carcinomas occurred in the dorsolateral and ventral lobes and were generally androgen receptor negative. Twenty-one of 32 (66%) mice aged > or = 6 months and 15 of 17 (88%) mice aged > or = 9 months developed metastatic tumors, as confirmed by histology and/or Tag immunohistochemistry. Metastases occurred at the later time points, with metastasis to regional lymph nodes, liver, and lung being particularly common. Metastases showed histological features of NE differentiation, as confirmed by chromogranin immunostaining and electron microscopy. An athymic nude mouse that received a s.c. implant of a primary NE tumor developed Tag-positive metastatic tumors with similar NE differentiation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified identical protein profiles between the primary NE tumor and lesions in the extraprostatic organs. Hence, in the 12T-10 large probasin promoter-Tag mouse, high-grade prostatic intraepithelial neoplasia develops progressively greater NE differentiation and progresses to invasive adenocarcinoma and NE carcinoma, with a high percentage of metastases. The predictable progression through these stages will allow testing of therapeutic interventions as well as possible further delineation of the role of NE cells in Pca progression.

Comparison of the mechanisms of two distinct aldolases from Escherichia coli grown on gluconeogenic substrates.

Escherichia coli grown on gluconeogenic compounds as carbon sources produced two chemically and physically distinct types of fructose-1,6-biphosphate aldolases (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphatelyase, EC 4.1.2.13), while these bacteria produced only a single enzyme when grown on glucose or fructose. We have investigated this enzyme in several strains of Escherichia coli (Crookes, K-12, and B) grown on glucose, fructose lactate, pyruvate, alanine and glycerol by comparing chemical properties and mechanisms of action. Comparison of these mechanisms was accomplished by following the fate of 18O in the keto position of fructose 1,6-bisphosphate during the aldolase catalyzed cleavage reaction. The results show that the two enzymes have different mechanisms of action and are consistent with a Schiff-base mechanism for the one which was induced by gluconeogenic substrates and metal-chelate mechanism for the constitutive enzyme.

Classification of fructose-1,6-bisphosphate aldolases based on 18O retention in the cleavage reaction.

Oxygen (18) was used as a mechanistic probe in the investigation of several different sources of fructose 1,6-bisphosphate aldolases (EC 4.1.2.13) which, due to differences in some physical and chemical properties, could not be clearly put in either Class I or Class II. Aldolases may be identified as belonging to a particular class on the basis of the amount of 180 retained in the dihydroxyacetone phosphate produced in the cleavage of [2-Oxygen (18)] fructose 1,6-biphosphate. The mechanism of Class I aldolases involves an obligatory exchange of the C-2 oxygen atom of fructose 1,6-bisphosphate, leading to the absence of 180 in the product. For Class II aldolases, the C-2 oxygen atom is retained in the aldol cleavage reaction. Aldolases from spinach and L. casei base intermediate. Aldosase from C. perfringens was found to be Class II, suggesting a metal-chelate intermediate. Results with Euglena aldolase confirmed that this organism contained both types of aldolases with approximately 78% Class II. The data show that despite a wide variety of physical and chemical properties, there are important mechanistic similarities within each class of enzyme and significant differences between the two classes. The determination of 180 retention in the product of the cleavage reaction using [2-180] fructose 1,6-biphosphate is an accurate means of classifying these enzymes since it is a measure of a property which is directly related to the mechanisms of the reactions.

Quantitation of plasma azathioprine and 6-mercaptopurine levels in renal transplant patients.

The plasma of renal transplant patients was analyzed by high performance liquid chromatography (HPLC) for the presence of azathioprine and its primary metabolite, 6-mercaptopurine, after either oral or i.v. administration of azathioprine. Azathioprine was demonstrated in plasma at peak concentrations of 0.6 microgram/ml 15 min after i.v. injections of 100 to 200 mg. Within 90 min of injection, the azathioprine level fell to 10 ng/ml. Azathioprine was not detected in plasma at any time after an oral dose of 100 mg, indicating that the plasma concentration is less than 0.5 ng/ml, which is the sensitivity limit of this assay. 6-Mercaptopurine appeared in the plasma after either oral or i.v. azathioprine administration. Furthermore, decreased renal graft function has no effect on the rate of disappearance of azathioprine from plasma. These results demonstrate that high performance liquid chromatography can be used to determine azathioprine and 6-mercaptopurine levels in man, and that alteration in renal function does not influence early stages of azathioprine degradation.

Relationship of human milk pH during course of lactation to concentrations of citrate and fatty acids.

Human milk pH was measured in 309 samples obtained from 52 women who had delivered at term and lactated for as long as 10 months thereafter. The mean pH decreased from 7.45 for colostrum to a nadir of 7.04 during the second week of lactation. Thereafter, the pH of milk remained between 7.0 and 7.1 until 3 months postpartum and then increased gradually to 7.4 by 10 months. The change in hydrogen ion concentration in milk was associated with corresponding changes throughout lactation in the concentration of citrate but not with the concentration of lactose. Lactose concentration increased gradually for 3 weeks; the concentration of saturated medium-chain fatty acids increased more rapidly. One interpretation of these findings is that the hydrogen ions and citrate generated by mammary secretory cell metabolism are used after the second week of lactation for de novo synthesis of fatty acids more rapidly than they are synthesized. Milk samples from ruminants were found to have concentrations of hydrogen ions and citrate that are greater than and pH that is less than the respective measurements in human milk. The significance for the recipient infant of the predictable changes in human milk pH during lactation and of the higher pH of human milk throughout lactation relative to bovine milk is unknown. However, drug excretion into milk, milk enzyme activity, milk leukocyte function, and neonatal gastrointestinal function are affected by ambient pH and may be influenced by the pH of milk.

Micro-electrospray mass spectrometry: Ultra-high-sensitivity analysis of peptides and proteins.

A "micro-electrospray" ionization source has been developed that markedly increases the sensitivity of the conventional electrospray source. This was achieved by optimization of the source to accommodate nanoliter flow rates from 300 to 800-nL/min spraying directly from a capillary needle that, for the analysis of peptides, contained C18 liquid chromatography packing as an integrated concentration-desalting device. Thus, a total of 1 fmol of methionine enkephalin was desorbed from the capillary column spray needle, loaded as a 10-µL injection of 100-amol/µL solution. The mass spectrum showed the [M + H](+) ion at m/z 574.2 with a signal-to-noise ratio of better than 5:1 from a chromatographic peak with a width of about 12 s. A narrow range (15-u) tandem mass spectrum was obtained for methionine enkephalin from the injection of 500 amol, and a full-scan tandem-mass spectrum was obtained from 50 fmol. For proteins, the average mass measurement accuracy was approximately 100-200 ppm for the injection of 2.5 fmol of apomyoglobin and 20-40 ppm for 200 fmol. Carbonic anhydrase B and bovine serum albumin showed similar mass measurement accuracies.

An integral probe for capillary zone electrophoresis/continuous-flow fast atom bombardment mass spectrometry.

An integral probe for capillary zone electrophoresis/continuous-flow fast atom bombardment mass spectrometry was constructed and operated in either the coaxial or liquid-junction interface mode. Results using these interfaces for the analyses of synthetic peptides are presented. The coaxial arrangement attains the best electrophoretic performance, generally providing a greater number of theoretical plates. However, in that the electrophoresis times are generally greater for the liquid-junction interface because there is no mechanical flow resulting from the source vacuum, the overall separation efficiency of the liquid-junction interface is equal to or greater than that of the coaxial interface. In addition, the liquid-junction interface is easier to set up and operate, and allows larger inner diameter capillaries to be used to achieve higher sample loads.

Automated mass spectrometry imaging with a matrix-assisted laser desorption ionization time-of-flight instrument.

The automated use of a matrix-assisted laser desorption ionization (MALDI) mass spectrometer (MS) is described for image analysis of samples through implementation of new software for instrument control, data acquisition, and data analysis. The software permits automated acquisition of MS MALDI spectra to form an ordered data array and contains display features to provide images at one or more mass-to-charge ratio values. The technique can be used to scan tissue samples, blotted samples, gels, or other sample surfaces where the image analysis of that sample is required. The program achieves a time of typically 1 s per image point, permitting an analysis made up of large numbers of points with high spatial resolution up to 850 dpi. The features of the software are demonstrated in this paper with samples of printed images, where visible images can be compared to those obtained by mass spectrometry. Quantitative aspects are introduced by analyzing a series of sample spots containing different amounts of several proteins.

Micro-Electrospray: Zeptomole/attomole per microliter sensitivity for peptides.

The micro-electrospray ionization source has been optimized for the specific analysis of neuropeptides such as neurotensin and methionine enkephalin. The source has the option of integrating nanoliter flow-rate desalting and preconcentration techniques into the micro-electrospray spray needle, eliminating post-column dead volumes. For neurotensin, the most sensitive neuropeptide analyzed thus far in this work, the injection of 10 µL of a solution containing 320 zeptomolesy/gmL gave an [M + 3H](+3) ion at m/z 558.4 with S/N of > 8∶1. The MS/MS analysis of this peptide for the fragment ion at m/z 578.9 gave a S/N > 20∶1 for a solution containing 32 attomoles/µL.

Capillary electrophoresis combined with matrix-assisted laser desorption/ionization mass spectrometry; continuous sample deposition on a matrix-precoated membrane target.

Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) were combined in an off-line arrangement to provide separation and mass analysis of peptide and protein mixtures in the attomole range. A membrane target, precoated with MALDI matrix, was used for the continuous deposition of effluent exiting from a CE device. A sample track was produced by linear movement of the target during the electrophoretic separation and this track was subsequently analyzed by MALDI/MS. The technique is effective for peptides and proteins, having limits of detection (signal-to-noise >3) of about 50 amol for neurotensin (1673 Da) and 250 amol for cytochrome c (12361 Da) and apomyoglobin (16951 Da). The electrophoretic separation achieved from the membrane target, as measured by theoretical plate numbers from the mass spectrometric data, can be as high as 80-90% of that achieved by on-line UV detection under optimal conditions, although band broadening occurs and with some loss of separation efficiency. Non-volatile buffers such as 10-50 mM phosphate can also be used in the electrophoresis process and directly deposited on the membrane. The use of post-source decay techniques is shown for peptides in the CE sample track in order to obtain sequence verification. The effectiveness of this method of integration of CE and MALDI/MS is demonstrated with both peptide and protein mixtures and with the analysis of a tryptic digest of a protein.

Determination of protein-protein interactions by matrix-assisted laser desorption/ionization mass spectrometry.

A number of different procedures have been developed for use with matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) for the analysis of non-covalent protein-protein complexes. These include use of specific matrix and laser combinations, accumulation of "first shot" spectra, modification of pH and solvent conditions during sample preparation and use of cross-linking agents to attach the monomers covalently to each other in the complex. The results have shown the techniques to be effective with some but not all complexes, although cross-linking is the most successful. The physical and chemical nature of the complex is critical and therefore a diversity of approaches is recommended for such studies.

Mapping of surrogate markers of cellular components and structures using laser desorption/ionization mass spectrometry.

Laser desorption/ionization mass spectrometry (LDI-MS) has been used to assess the potential of using surrogate markers, bound to cellular structures containing nucleic acids, to image or map the position of these structures within biological samples. In this study, organic dyes were used as markers because of their established use in the histochemical marking of nucleic acids, and also because they are amenable to LDI-MS. Eight cationic dyes were tested and all could be desorbed from nucleic acid samples without additional matrix after specifically binding to these molecules. Methylene Blue was the best of these based on its sensitivity to detection by LDI-MS and the fact that it can be washed from the tissue in areas where it was not specifically bound to provide low-intensity background signals. Experiments are reported which characterize the M(+) ion signal obtained from Methylene Blue with regard to sensitivity, reproducibility and possible use for quantitation. This dye was used to map (with a lateral resolution of 25 microm) several nucleic acid-containing samples spotted on prepared surfaces, and to image the location of nucleic acids in two model tissues, retinal vertical sections and thyroid whole mount sections.