Membrane mobility agent alters the consequences of lectin-cell interaction in a malignant cell membrane

The membrane mobility agent 2-(2-methoxyethoxy)-ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate promotes cap formation from wheat germ agglutinin-receptor combinations at the expense of agglutination in membranes of malignant mastocytoma cells.     

The mechanism of wheat germ agglutinin mediated cytolysis of murine mastocytoma cells

The present study was undertaken to test whether cytolysis by wheat germ agglutinin requires lateral mobility of membranal lectin receptor sites into caps.Preincubation of interphase murine mastocytoma cells with 100 μg/ml trypsin promoted cap formation by the agglutinin in about 30% of the cells, followed by cytolysis of these cells. Pretreatment of the cells with NaN₃, low temperature or glutaraldehyde decreased degree of capping and to some extent decreased the degree of cytolusis, while the addition of antibodies to the agglutinin increased the degree of capping and lysis. A linear relationship with a high correlation coefficient exists between the degree of capping and the degree of cytolysis, suggesting that lateral mobility of membrane wheat germ agglutinin receptors is required for cytolysis by the lectin. Further studies have shown the restricted small hole damage followed by osmotic lysis is responsible for the damage induced by the agglutinin of trypsin-treated mastocytoma cells. This was demonstrated (a) by markers of low molecular weight (⁸⁶Rb) which were released from the cells before those of high molecular weight (⁵¹Cr-protein) and (b) by protecting the cells from lysis through incubating them in non-penetrating solutes, such as Dextrans of high molecular weight. It has been calculated the the initial size of the lytic lesion induced by wheat germ agglutinin is $\text{≈ 32}\text{A}\text{?}$.     

Nephritogenoside,the receptor glycoprotein for concanavalin a in rat glomerular basement membrane: Demonstration of α-glucopyranosyl unit at the non-reducing terminus

Rat glomerular basement membrane was extracted for 3 h with trypsin, pH 8.0. The supernatant solution was treated with trichloroacetic acid and the supernatant thus obtained was applied to Bio-Gel P200. The first of the two glycoprotein peaks was applied onto Sepharose derivatives of concanavalin A (Con A.).Examination of the material retained by the unsolubilized Con A and subsquently eluted with methyl α-d-mannopyranoside reveals that the principal high affinity receptor for Con A is the renal glycoprotein, having antigenic activity that induces nephrotoxic antibody. This glycoprotein has also nephritogenicity (the activity capable of inducing glomerulonephritis in homologous animals by a single foot pad injection with Freund's incomplete adjuvant). Evidence is given to show that this binding is specific.The remainder of the renal glycoprotein is unretarded and is revealed to contain none of the activities described above.When fluorescein isothiocyanate-labelled Con A is, conversely, injected into rats through the renal artery, the specific binding of Con A to the glomerular capillary loop is proved.The results demonstrated here appear to, indicate that the receptor for Con A present in normal rat glomerular basement membrane can be identified as the well-established chemical substance, the nephritogenoside, having the α-d-glucopyranosyl unit at the non-reducing terminus which is facing the endothelial aspects of the glomerular capillary loop.     

D-glucosyl isothiocyanate, an affinity label for the glucose transport proteins of the human erythrocyte membrane

D-glucosyl isothiocyanate has been found to be a potent irreversible inhibitor of glucose translocation in the human erythrocyte. [¹⁴C]-D-glucosyl isothiocyanate was incorporated into two major proteins of the erythrocyte membrane, and this incorporation was inhibited by D-glucose. These proteins may be components of the glucose transport system.     

Rotational diffusion of Escherichia coli ribosomes. I. - Free 70 S, 50 S and 30 S particles.

The rotational brownian diffusion of Escherichia coli ribosomes has been studied by following the transient dichroism generated by optical excitation of a covalent probe into its triplet state. The induced absorption anisotropy decays exponentially with characteristic correlation times: 2.5 microseconds, 1.6 microseconds and 1.1 microseconds for the 70S ribosome and the 50S and 30S subparticles respectively. The corresponding Stokes radii are in the same order, 133 A, 115 A and 103 A. The hydrodynamic properties are discussed in terms of an ellipsoidal shape of the ribosome particles.     

Changes in subcellular localization of fructose 1,6-bisphosphatase during differentiation of isolated muscle satellite cells

Subcellular localization of FBPase, a regulatory enzyme of glyconeogenesis, was examined inside dividing and differentiating satellite cells from rat muscle. In dividing myoblasts, FBPase was located in cytosol and nuclei. When divisions ceased, FBPase became restricted to the cytosolic compartment and finally was found to associate with the Z-lines, as in adult muscle. Moreover, a 12-fold decrease was observed in the number of FBPase-positive nuclei associated with muscle fibres of adult rat, as compared with young muscle, possibly reflecting the reduction in number of active satellite cells during muscle maturation. The data might suggest that FBPase participates in some nuclear processes during development and regeneration of skeletal muscle.     

Induction of auto-logous human cytotoxic T lymphocytes (CTL) from peripheral blood against tumor cells

Synthetic 4-methylsulfinylhexyl isothiocyanate (MITC)(a potent inducer of phase 2 detoxification enzymes from broccoli) and 6-MITC(a potent anti-proliferative principal from wasabi) slightly inhibited the induction of mouse skin tumor in a two-stage process of carcinogenesis (initiator, 9,10-dimethyl-1,2-benzanthracene; promotor,12-o-tetradecanoylphorbol-13-acetate), but the effect was not significant. Both compounds, however, significantly inhibited the mutation of skin resulting from topical applications of the carcinogens. When a murine hepatoma cell line, Hepa 1c1c7, was treated with 2-,4-,6- and 8-MITCs, they augmented the induction of its quinone reductase, one of the phase 2 detoxification enzymes in a concentration dependent manner, and the 4- and 6-MITCs were much more potent on the reduction of the enzyme than the 2- and 8-MITCs. All 2-, 4-, 6- and 8-MITCs suppressed the growth of murine tumor cells, their suppressive activities being proportional to the length of their methyl residue. They were also cytotoxic to mouse peritoneal exudate macrophages which were not proliferating in vitro, indicating that the cellular targets of isothiocyanate may not be dependent upon the cell cycle. In addition, all the 2-, 4-, 6- and 8-MITCs inhibited the production of nitric oxide (a potent radical carcinogen) by peritoneal macrophages. This revised version was published online in June 2006 with corrections to the Cover Date.     

Specific Antimicrobial Synergism of Synthetic Hydroxy Isothiocyanates with Aminoglycoside Antibiotics

Hydroxy isothiocyanates, especially 2-(4-hydroxy-phenyl)ethyl isothiocyanate (hITC), were examined for antimicrobial synergism with several antibiotics against Escherichia coli and Staphylococcus aureus, using a multiwell plate system. hITC had antibacterial synergism, specifically with aminoglycoside antibiotics. The synergism was observed in synthetic medium (M9 minimal medium) or soybean casein digest broth, but not in nutrient broth. Synergism was seen in the presence of certain sugars such as glucose, fructose, and maltose in the medium.     

Antifungal Vapour‐phase Activity of a Combination of Allyl Isothiocyanate and Ethyl Isothiocyanate Against Botrytis cinerea and Penicillium expansum Infection on Apples

The potential use of allyl isothiocyanate (AITC) and ethyl isothiocyanate (EITC), singly and in combination, was tested in in vitro and in vivo trials for their effect on Penicillium expansum Link and Botrytis cinerea Persl. infection on apple when used as a fumigant. A 3 : 1 ratio of AITC : EITC was more efficient at reducing in vitro spore germination of P. expansum and B. cinerea than were other combinations or either AITC or EITC alone. The optimized combination showed the lowest EC₅₀ values, at 0.08 and 0.14 μg/ml air, for P. expansum conidial germination and mycelial growth, respectively, and 0.07 and 0.12 μg/ml air for B. cinerea conidial germination and mycelial growth, respectively. In in vivo trials, artificially infected apples were exposed for 4 days to an ITC‐enriched atmosphere. Among the ITCs tested, AITC, EITC and their combinations reduced incidence by more than 85% after 3–4 days of apple incubation at 20°C. Although further studies are necessary to evaluate any detrimental effects on apple quality, the evidence from this study supports the use of fumigation based on ITCs, and in particular a 3 : 1 combination of AITC and EITC, for control of postharvest mildew in apple fruit.     

Effect of Synthetic Hydroxy Isothiocyanates on a Bacterial Virus and DNA

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on φK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.     

Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase

Fluorescein isothiocyanate was used to covalently label the gastric (H⁺ + K⁺)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated $\text{p-}\text{nitrophenylphosphatase}$ activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of $\text{p}\text{I}$ 5.6 was also noted. Fluorescence was quenched by K⁺, Rb⁺ and Tl⁺ in a dose-dependent manner, and the $\text{K}{}_{0.5}$ values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg²⁺-dependent fluorescent quench. Ca²⁺ reversed the K⁺-dependent loss of fluorescence and inhibited it when added prior to K⁺. This may relate to the slow phosphorylation in the presence of ATP found when Ca²⁺ was substituted for Mg²⁺ and the absence of K⁺-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K⁺ binding to the enzyme.     

1-cyanoepithioalkanes: Major products of alkenylglucosinolate hydrolysis in certain cruciferae

Autolysis of glucosinolates in several crucifers produced 1-cyano-2,3-epithio-propane and 1-cyano-3,4-epithiopentane, in addition to the previously described 1-cyano-3,4-epithiobutane.     

Chemical alterations and regulations of biomolecules in lifestyle-related diseases

We know experientially that not only nutrient factors but also non-nutritive functional food factors are playing important roles in maintenance of homeostasis, health promotion, and disease prevention. Although some of these effective behaviors are supported by accumulating scientific evidences, it is in general difficult to determine properly in human. Therefore, the discovering of novel biomarker and developments of the analytical method are one of the prudent strategies to understand disease etiology and evaluate efficacies of functional food factors via monitoring the pathophysiological alteration in live body, tissue, and cells. This review describes recent our findings on (1) formation mechanism, bioactivities, quantitative determination of cholesterol ozonolysis product, secosterol as possible biomarker for lifestyle-related disease, and (2) chemical biology approach for the investigating molecular mechanisms of most promising cancer chemopreventive food factors, isothiocyanate-inducing bioactivities.     

Efficient and environmentally friendly method for carotenoid extraction from Paracoccus carotinifaciens utilizing naturally occurring Z-isomerization-accelerating catalysts

This study aimed to improve the efficiency of Paracoccus carotinifaciens-derived carotenoid (astaxanthin, adonirubin, adonixanthin) extraction using environmentally friendly Z-isomerization-accelerating catalysts. Adding naturally occurring catalysts such as isothiocyanates and polysulfides to the extraction solvent significantly improved the efficiency of carotenoid extraction, likely because of enhanced solubility of carotenoid Z-isomers compared with all-E-isomers. Indeed, addition of the catalysts markedly increased the content of carotenoid Z-isomers in the resulting extract. The use of a catalyst allyl isothiocyanate, which is abundantly included in Brassicaceae plant family, at high extraction temperature and long extraction time led to increased carotenoid recovery and Z-isomer content. These findings will enhance the efficiency of organic solvent-based extraction of carotenoids from carotenoid-rich sources. Numerous studies have reported that the Z-isomers of carotenoids exhibit greater bioavailability and antioxidant capacity than the all-E-isomers. Hence, the method proposed here utilizing Z-isomerization-accelerating catalysts could enhance both the extraction efficiency and beneficial health effects of carotenoids.     

Automated carboxy-terminal sequence analysis of peptides.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-terminal sequence analysis of peptides.     

Automated large-volume sample stacking procedure to detect labeled peptides at picomolar concentration using capillary electrophoresis and laser-induced fluorescence detection

We have developed an automated large-volume sample stacking (LVSS) procedure to detect fluorescein isothiocyanate-labeled peptides in the picomolar range. The injection duration is 10 min at 50 mbar to fill 62% of the capillary volume to the detection cell. The calculated limit of detection (S/N=3), filling 1% of the capillary volume, is 74 pM for bradykinin and 45 pM for L-enkephalin with samples diluted in water and analyzed in a 50 mM borate buffer, pH 9.2. With the automated LVSS system, the limits of detection are 7 pM for bradykinin, 3 pM for L-enkephalin and 2 pM for substance P. LVSS is shown to be quantitative from 500 to 10 pM.     

The reaction of fluorescein isothiocyanate with thiols: a method for assay of isothiocyanates

The reversible formation of dithiourethanes from fluorescein isothiocyanate and mercaptoethanol or mercaptoethylamine has been studied. It is shown that it is possible to use the reaction as the basis for a spectrophotometric titration of a number of isothiocyanates with mercaptoethanol.     

Improved initial yields in C-terminal sequence analysis by thiohydantoin chemistry using purified diphenylphosphoryl isothiocyanate: NMR evidence for a reaction intermediate in the coupling reaction

A thiohydantoin method for C-terminal sequence analysis of proteins on Zitex membranes involves derivatization of the free alpha-carboxyl group with diphenylphosphoryl isothiocyanate (DPPITC) plus treatment with pyridine to form a peptidylthiohydantoin derivative, cleavage of the thiohydantoin (TH) amino acid from the protein with potassium trimethylsilanolate, and identification of the released TH-amino acid by online reversed-phase HPLC. This automated chemistry, which was adapted to the Hewlett-Packard G 1009A sequencer, has been shown to identify two or three cycles on a wide variety of proteins, but suffers from low initial yields and instability of the DPPITC reagent. We report here an improved method for synthesis and purification of DPPITC. With this procedure the DPPITC reagent is a clear liquid that is stable at room temperature under vacuum for more than 9 months or for more than 24 months as a 1.0M solution in benzene at -20 degrees C. Using the purified reagent we were able to more than double the initial yield (from 30.7 to 72.4%) of TH-amino acid from a test protein and substantially decrease sequencer background. Examination of the reaction between DPPITC and the carboxylate of model N-terminally protected dipeptides with 31P NMR provides spectroscopic evidence for a postulated intermediate formed between the DPPITC and the peptide carboxylate. The reaction intermediate provides new insight into the coupling mechanism.