Subcellular localization of two neurotropic drugs in three varieties of central nervous system cells by secondary ion mass spectroscopy (SIMS) microscopy.

The intracellular localization of two neurotropic drugs, flunitrazepam (benzodiazepine) and triflupromazine (phenothiazine), was studied by secondary ion mass spectrometry microscopy (SIMS) in three varieties of cells. The images of the intracellular distributions of the two drugs are easily obtained by selecting the fluorine atom of the molecules. These images show that the drug from the benzodiazepine group is mainly located in the nuclei, whereas the phenothiazine is exclusively located inside the cytoplasm.     

The composition and structure of bovine peritubular dentin: mapping by time of flight secondary ion mass spectroscopy

The dentin layer of the tooth is a complex mineralized tissue traversed by a closely packed system of tubules. Each tubule is surrounded by highly mineralized tissue referred to as peritubular dentin (PTD). The remaining mineralized collagen network between the tubules is the intertubular dentin (ITD). A TOF-SIMS analysis of the PTD constituents has been used to compare the PTD to the ITD. The PTD differs from the ITD not only in the degree of mineralization but also in the amount and nature of the mineral elements and amino acids. The organic matrix of the PTD consists of a unique collagen free assembly of proteins rich in glutamic acid, where the ITD organic matrix is collagen-rich and Asp-rich. The apparent concentration of organic fragment ions observed in the PTD in the TOF-SIMS negative ion mode was much higher than expected. The PTD was found to be rich in Ca2+, Na+, Mg2+, and K+. The amount of Mg2+ and K+ in the PTD was significantly reduced after deproteination, while Ca2+ and Na+ were still accumulated in the PTD. This implies that Mg2+ and K+ are mainly associated with the organic matrix rather than with the mineral of the PTD.     

The role of secondary ion mass spectrometry (SIMS) in biological microanalysis: technique comparisons and prospects.

The virtues and limitations of SIMS ion microscopy are compared with other spectroscopic techniques applicable to biological microanalysis, with a special emphasis on techniques for elemental localization in biological tissue (electron, X-ray, laser, nuclear, ion microprobes). Principal advantages of SIMS include high detection sensitivity, high depth resolution, isotope specificity, and possibilities for three-dimensional imaging. Current limitations, especially in comparison to X-ray microanalysis, center on lateral spatial resolution and quantification. Recent SIMS instrumentation advances involving field emission liquid metal ion sources and laser post-ionization will help to minimize these limitations in the future. The molecular surface analysis capabilities of static SIMS, especially with the new developments in commercial time-of-flight spectrometers, are promising for application to biomimetic, biomaterials, and biological tissue or cell surfaces. However, the direct microchemical imaging of biomolecules in tissue samples using SIMS will be hindered by limited concentrations, small analytical volumes, and the inefficiencies of converting surface molecules to structurally significant gas phase ions. Indirect detection using elemental or isotopically tagged molecules, however, shows considerable promise for molecular imaging studies using SIMS ion microscopy.     

Comparative study of acidic glycosphingolipids by field desorption and secondary ion mass spectrometry

Acidic glycosphingolipids were analyzed by field desorption (FD-MS) and secondary ion mass spectrometry (SI-MS) using the primary ion Xe+ with a glycerol matrix. In the analysis of underivatized gangliosides by FD-MS, the fragment corresponding to the asialo residue resulting from the cationized cluster ion (M + Na)+ was the base peak, and ions due to cleavage at the glycosidic linkages were detected, as in the neutral glycosphingolipids. In the case of sulfatide, the ceramide fragment showed the highest intensity in the spectrum. In SI-MS spectra of acidic glycosphingolipids, (M + Na)+, (M + 2Na-H)+, and (M + K)+ were continuously detected as relatively high intensity ions during analysis of gangliosides and sulfatide. Other ions were mostly similar to those obtained by FD-MS. In FD-MS spectra of permethylated gangliosides, the cationized molecular ion (M + Na)+ was the base peak, and fragment ions due to asialo gangliosides were prominent. Other peaks were hard to detect. In SI-MS, molecular ions (M + H)+ and (M + H-32)+ and other ions due to cleavage of the glycosidic linkages were clearly detected. In this case, the sensitivity was greatly improved. Ions due to the non reducing end sugars were clearly detected, because of the relatively low intensity of ion peaks due to the glycerol matrix. It is concluded that the combination with FD-MS and SI-MS is particularly useful for the determination of molecular weight, sugar sequence and ceramide structure with sample amounting to only a few micrograms order.     

Orientation of membrane-bound melittin studied by a combination of HPLC and liquid secondary ion mass spectrometry (LSIMS)

Combining two analytical techniques, HPLC and liquid secondary ion mass spectrometry, the orientation of liposomal membrane-bound melittin was analyzed through its trypsin-digested products. We found that trypsin can access all proteolytic sites of the membrane-bound melittin when the liposomes have no transmembrane potential, whereas the proteolytic site near the N terminus of melittin is blocked when the liposomes have a negative transmembrane potential. The results suggest that the negative transmembrane potential may induce the melittin molecules to insert into the membrane perpendicularly, whereas melittin lies flat on the membrane surface in the absence of a negative potential.     

The use of secondary ion mass spectrometry (SIMS) to evaluate internal contamination.

Secondary ion mass spectrometry (SIMS) permits the detection of stable and radioactive elements in microvolume. Based on the ablation of specimens by ion bombardment, this mass spectrometry method allows a rapid assessment of trace elements in biological samples and enables accurate isotopic ratio determination. In this work, an application of SIMS in studies involving element microdistribution is illustrated on the basis of analyses of duodenal tissue sections from rats contaminated with either cerium or thorium. For this purpose, tests are performed with SIMS to analyze tissue sections obtained 12, 24 and 48 hr after contamination. In this report, strengths and limitations of SIMS are pointed out as an important tool in biological research.     

4-氨基苯酚维甲酰胺对肝癌和恶性黑色素瘤细胞的抑制作用

In this paper, a significantly effect of N-(4-hydrophenyl) retinamide (4-HPR), a derivative of retinoic acid, was observed on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatoma cells and B16 melanoma cells. The number of migratory hepatoma cells reduced significantly from the control 201 +/- 27.2 to 58 +/- 5.03 after 6-hour incubation with 4-HPR (p < 0.01, n = 4). The number of migratory B16 melanoma cells reduced from the control 302 +/- 30.1 to 254 +/- 25.04 (p < 0.05, n = 4). The invasive ability of these cells was also suppressed by 4-HPR treatment. Cells that penetrated the artificial membrane matrigel decreased from 27 +/- 13.1 to 11.2 +/- 3.3 in hepatoma cells, from 67.5 +/- 10.1 to 24.3 +/- 3.2 in B16 melanoma cells (p < 0.05, n = 3). Furthermore, cell growth was significantly inhibited especially in B16 melanoma cells and 37.11 +/- 0.94% cells were induced to apoptosis after 48-hour induction by 4-HPR, which was significantly higher than those by retinoic acid treatment (p < 0.05). Although the mechanism of 4-HPR effects was not very clear, over expression of CST, which was inhibited by 4-HPR in our previous study, could diminish the apoptosis--inducing effect by 4-HPR. We believe that 4-HPR has a strong inhibitory effect on melanoma and hepatocarcinoma cells and might become a potent therapeutic agent.     

H assignment and secondary structure determination of human melanoma growth stimulating activity (MGSA) by NMR spectroscopy

The solution structure of melanoma growth stimulating activity (MGSA) has been investigated using proton NMR spectroscopy. Sequential resonance assignments have been carried out, and elements of secondary structure have been identified on the basis of NOE, coupling constant, chemical shift, and amide proton exchange data. Long-range NOEs have established that MGSA is a dimer in solution. The secondary structure and dimer interface of MGSA appear to be similar to those found previously for the homologous chemokine interleukin-8 [Clore et al. (1990) Biochemistry 29, 1689-1696]. The MGSA monomer contains a three stranded anti-parallel β-sheet arranged in a ‘Greek-key’ conformation, and a C-terininal -helix (residues 58 69).     

Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy)

This paper reviews the most recent methodological advances in the field of biological imaging using dynamic secondary ion mass spectrometry (SIMS). After a short reminder of the basic principle of SIMS imaging, the latest high-resolution dynamic SIMS equipment is briefly described. This new ion nanoprobe (CAMECA NanoSIMS 50) has a lateral resolution of less than 50 nm with primary Cs+ ion, the ability to detect simultaneously 5 different ions from the same micro-volume and a very good transmission even at high mass resolution (60% at M/DeltaM=5000). Basic considerations related to sample preparation, mass resolution and primary ion implantation are given. The decisive capability of this new instrument, and more generally of high-resolution dynamic SIMS imaging in biology, are illustrated with the most recent examples of utilization.     

The site of action of lithium ions in morphogenesis of Lymnaea stagnalis analyzed by secondary ion mass spectroscopy

Abstract. Exogastrulation as a disturbance of development in eggs of Lymnaea stagnalis is caused by the action of LiCl at the second cleavage stage and not at the first or third. The percentage of exogastrulae formed is strongly concentration dependent. To determine the site of action of lithium ions, the cellular contents of Li, C, Na, Mg, P, K, and Ca were analyzed by secondary ion mass spectroscopy (SIMS). The mean elemental concentrations of Na, Mg, K, and Ca are close to those found earlier by electron probe microanalysis and atomic absorption spectroscopy. Lymnaea eggs at the first, second, and third cleavage stage were treated with LiCl in a series of concentrations ranging from 50 to 0.1 mM. In all cases the cells contained a few mM lithium after treatment. After treatment at the insensitive first cleavage stage the lithium content is carried over by the cells through the sensitive second cleavage to the insensitive third cleavage stage. These data allow the conclusion that it is the external lithium concentration which is responsible for the specific effect. This presents direct analytical evidence that the primary action of lithium ions is located at the cell membrane.     

Theoretical study of the experimental coordination behavior of N-(2-aminophenyl)-D-glycero-D-gulo-heptonamide to Hg(II) ion

The reactivity of N-(2-aminophenyl)-d-glycero-d-gulo-heptonamide (adgha), with the group 12 cations, Zn(II), Cd(II), and Hg(II), was studied in DMSO-d₆ solution. The studied system showed a selective coordination to Hg(II), and the products formed were characterized by ¹H and ¹³C NMR in DMSO-d₆ solution and fast atom bombardment (FAB⁺) mass spectra. The expected coordination compounds, [Hg(adgha)](NO₃)₂ and [Hg(adgha)₂](NO₃)₂, were observed as unstable intermediates that decompose to bis-[2-(d-glycero-d-gulo-hexahydroxyhexyl)-benzimidazole-κN]mercury(II) dinitrate, [Hg(ghbz)₂](NO₃)₂. The chemical transformation of the complexes was followed by NMR experiments, and the nature of the species formed is sustained by a theoretical study done using DFT methodology. From this study, we propose the structure of the complexes formed in solution, the relative stability of the species formed, and the possible role of the solvent in the observed transformations.     

Sample preparation of animal tissues and cell cultures for secondary ion mass spectrometry (SIMS) microscopy.

Sample preparation is a critical step in the elemental analysis of animal tissues and cell cultures with ion microscopy. Since live cells cannot be analyzed with ion microscopy, a careful sample fixation is necessary which preserves the native structural and chemical integrity of a specimen. The evaluation of morphological and chemical integrity of a fixed specimen is necessary before any physiological explanation of ion fluxes is interpreted based on ion microscopy. For diffusible ion localization studies, strict cryogenic procedures are recommended. Examples are shown for diffusible ion microanalysis in frozen-freeze-dried tissues and cell cultures. Ion microscopy studies of tightly bound elements/molecules may be conducted in chemically fixed and/or plastic embedded specimens. Since it is not generally known which elements/molecules are tightly bound to the tissue matrix, a confirmation of elemental distribution with cryogenic procedures is desirable. A recent approach of combining laser scanning confocal fluorescence microscopy and ion microscopy on the same frozen freeze-dried cell is also discussed for recognizing smaller cytoplasmic structures in ion microscopy images.     

N-(4-hydroxyphenyl)retinamide (Fenretinide) and nephrectomy alter normal plasma retinol-binding protein metabolism

We have reported previously that injecting vitamin A-deficient rats with N-(4-hydroxyphenyl)retinamide causes a significant reduction in the liver retinol-binding protein concentration and a 2 fold rise in the kidney retinol-binding protein concentration. This presumably reflects a rapid translocation of retinol-binding protein from the liver to the kidney through the plasma, although no rise in plasma retinol-binding protein is detected. In the present studies, nephrectomized rats were used to determine if retinol-binding protein accumulating in kidneys passes through the plasma. Bilateral nephrectomy in control rats caused the plasma retinol-binding protein concentration to approximately double by 5 hr postsurgery. However, nephrectomy plus N-(4-hydroxyphenyl)retinamide treatment did not result in an increase in the plasma retinol-binding protein concentration. Therefore, the lowering of liver retinol-binding protein concentration in response to N-(4-hydroxyphenyl)retinamide treatment was not accounted for by an accumulation of retinol-binding protein in the plasma compartment. Interestingly, the muscle retinol-binding protein concentration increased with nephrectomy plus N-(4-hydroxyphenyl)retinamide treatment. The ratio of muscle retinol-binding protein:plasma retinol-binding protein in vitamin A-deficient nephrectomized rats treated with N-(4-hydroxyphenyl)retinamide was significantly higher than in comparable rats treated with either carrier or retinol. We conclude that in vivo N-(4-hydroxyphenyl)retinamide induces the secretion of retinol-binding protein from the liver. Since the N-(4-hydroxyphenyl)retinamide-retinol-binding protein complex does not bind with transthyretin it rapidly leaves the plasma. In non-nephrectomized rats this complex is rapidly filtered by the kidney. Nephrectomizing rats causes the retinol-binding protein secreted in response to N-(4-hydroxyphenyl)retinamide to diffuse into interstitial fluid.     

Mapping of magnesium and of different protein fragments in sea urchin teeth via secondary ion mass spectroscopy

Mature portions of sea urchin are comprised of a complex array of reinforcing elements yet are single crystals of high and very high Mg calcite. How a relatively poor structural material (calcite) can produce mechanically competent structures is of great interest. In teeth of the sea urchin Lytechinus variegatus, we recorded high-resolution secondary ion mass spectrometry (SIMS) maps of Mg, Ca ,and specific amino acid fragments of mineral-related proteins including aspartic acid (Asp). SIMS revealed strong colocalization of Asp residues with very high Mg. Demineralized specimens showed serine localization on membranes between crystal elements and reduced Mg and aspartic acid signals, further emphasizing colocalization of very high Mg with ready soluble Asp-rich protein(s). The association of Asp with nonequilibrium, very high magnesium calcite provides insight to the makeup of the macromolecules involved in the growth of two different composition calcites and the fundamental process of biomineralization.     

Quantitative study of secondary structure of histones H1, H2A, and H4 in solution by infrared spectroscopy.

The secondary structure of histones H1, H2A, and H4 (F1, F2a2, and F2a1) has been quantitatively studied in heavy water (2H2O) solutions in a wide range of histone concentration, p2H, and concentration of sodium chloride using an improved infrared spectroscopy method. Under all conditions there are about 5--10% of alpha helix. Conditions favourable for aggregation induce formation of antiparallel pleated sheet structure to an extent of about 15% in H1 and H2A and about 30% in H4. When the p2H and concentration of NaCl are in the physiological range, there is the same content of this structure in H2A and H4 and none in H1.     

Some features of hydrogen (ion) secretion by the frog skin.

We have studied the movements of H+ from the in vitro frog skin into the outside solution because it has been suggested that the movement of sodium from the outside solution into the skin may result from the forced exchange of Na+ by H+. Our main observations can be summarized as follows: (a) Hydrogen moves from the skin into the outside solution at a rate of 0.04 muequiv-cm-2-h-1 while Na+ influx had a value of 0.49 muequiv-cm-2-h-1. (b) The rate of H+ secretion is not significantly affected by substituting the Na+ in the outside solution by K+ nor by inhibiting Na+ influx with amiloride (5-10(-5) M). (c) Acetazolamide (5-10(-3) M) blocked H+ secretion without altering the potential difference across the skin. (d) The rate of H+ production is not underestimated because it may have been neutralized by HCO3- secreted into the outside solution in exchange for Cl-. Substituting all the Cl- by SO4(2-) in the outside solutions does not result in an increase in the rate of H+ production. (e) The steady-state rate of H+ secretion is not affected by large changes in electrochemical potential gradients for H+. Neither abolishing the potential difference across the skin nor a 10-fold change in H+ concentration in the outside solution affected significantly the steady-state rate of H+ secretion. (f) The H+ secretion was abolished by the metabolic inhibitors dinitrophenol (1-10(-4) M) and Antimycin A (1.5-10(-6) M) which also markedly reduced the potential difference across the skin. Observations (a), (b), and (c) suggest that H+ and Na+ movements across the outer border of the isolated frog skin are not coupled. The ratio of Na+ to H+ movements is very different from unity and Na+ movements can be abolished without any effects on H+ secretion and conversely H+ movements can be abolished without interruption of Na+ uptake. A second conclusion suggested by these results is that the H+ secretion does not result from movement of H+ following its electrochemical potential gradient since that rate of secretion is not affected by marked changes in either potential or [H+]. Furthermore, the effects of metabolic inhibitors suggest that H+ secretion requires the expenditure of energy by the cell.     

Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis.

A procedure for the synthesis of the fluorogenic substrate analogue 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid for the human acid neuraminidase has been developed. The substrate was employed for the characterization of the enzyme in sonicates of cultured human skin fibroblasts and for enzymatic detection of the neuraminidase deficiency in the neurological storage disorder, sialidosis. Synthesis was accomplished by reacting 2-deoxy-2-chloro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester with the sodium salt of 4-methylumbelliferone in acetonitrile at room temperature. The coupled product was purified on silicic acid chromatography, followed by base-catalyzed removal of the O-acetyl and methoxy blocking groups, and with additional purification of the hydrolyzed product on silicic acid. The overall yield, based on N-acetylneuraminic acid, was 37%. Under linear assay conditions, at pH 4.3, the apparent maximal velocities (nmol (mg of protein)-1 h-1) for normal fibroblasts were 58--115, 0.2--1.8 for sialidosis fibroblasts, and 28--38 for obligate heterozygotes. The apparent Km for normals was 0.13 mM.     

N-(2-hydroxy phenyl) acetamide: a novel suppressor of Toll-like receptors (TLR-2 and TLR-4) in adjuvant-induced arthritic rats

This study investigated the roles of Ras, ERK, and Akt in the glucocorticoid-induced differentiation of growth hormone-producing pituitary cells in vitro. Pituicytes isolated from day-18 rat embryos were cultured with 50 mM dexamethasone in addition to specific inhibitors of Ras (manumycin; 0.5, 5, 50 nM), ERK (U0126, 10 μM), or Akt (LY294002, 25 μM). Differentiation was assessed using immunofluorescent staining of intracellular growth hormone. Radioimmunoassay and Western blot analyses were used to determine levels of secreted and intracellular growth hormone, respectively. Manumycin reduced the fraction of growth hormone-positive cells and dexamethasone-induced growth hormone secretion in a dose-dependent manner (both P < 0.001). In the absence of dexamethasone, LY294002 and U0126 did not alter the fraction of growth hormone-positive cells or intracellular growth hormone protein expression or secretion. Both LY294002 and U0126 alone significantly attenuated the fraction of dexamethasone-treated GH-positive cells and the secretion of GH compared to those of cells treated only with dexamethasone (50 nM for 44 h or 48 h) (all P < 0.05). Dexamethasone treatment alone did not change GH protein levels. Treatment of cells with a combination of LY294402 and U0126 significantly attenuated the fraction of dexamethasone-treated GH-positive cells, GH protein levels, and GH secretion compared to cells treated with dexamethasone alone (all P < 0.05). Moreover, dexamethasone-induced phosphorylation of GTP-Ras, ERK, and Akt was significantly attenuated by exposure to the respective inhibitors (P < 0.05). Taken together, our results indicate that Ras, ERK, and Akt are key effectors in the glucocorticoid-induced differentiation of growth hormone-secreting cells.