Spermidine, analysis

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

Hildrum, K., Scanlan, R.A. and Libbey, L.M. (1976). Nitrosamines from the nitrosation of spermidine and spermine, in Walker, E., Bogovski, P. and Griciute. L., Eds., Environmental N-Nitroso Compounds Analysis and Formation, Polytechnical Institute Tallinn, Estonia. 

Figure 11.9 Schematic view of the experimental strategy for carrying out poly(Phe) synthesis in POPC liposomes, (i) Freeze-thaw (x 7) solution containing t-RNAP , poly(U), Phe, ATP, GTP, Mg(OAc)2, NH4CI, spermine, spermidine, phos-phoenolpyruvate. (ii) Soution containing pyruvate kinase, 100 000 g supernatant enzymes, 308 and SOS ribosomal subunits, (iii) 1. Free-thaw (x3) 2. Brief extrusion 3. Addition of EDTA (final concentration = 35 mM. (iv) Withdrawl of aliquots at indicated time and cold TCA precipitation. Analysis of the radioactivity remaining on the glass filter by p-scintillation counting. (Modified from Oberholzer et al, 1999.)...

A mixture of 1 (200 mg, 0.45 mmol) and spermidine 4b (380 mg, 1.1 mmol) in THE (10 mL) was reacted with NaBH(OEh)3 (2 equiv) at room temperature under an argon atmosphere. The reaction was monitored by TLC analysis. After the reaction was complete, the solvent was removed, the residue extracted with ethyl acetate, and then the combined organic extracts were dried over anhydrous Na2S04, before being concentrated. 

A systematic analysis to determine the biophysical characteristics of some cationic lipid/DNA complexes from this series was undertaken (Eastman et al., 1997). Product 1 (Figure 15.7) is currently being used in clinical trials as a carrier of CFTR gene in cystic fibrosis. The synthesis of these cholesteryl derivatives is similar to that of DC-Chol cholesteryl chloroformate is reacted with spermine or spermidine as polycation (Lee et al., 1996). 

Used to introduce chromophores into amines aids in resolution of putrescine, spermidine, and spermine by HPLC excess TsCI must be removed (by extraction with hexane, for example) before analysis Reference 41... 

As for deaminase, the kinetic analysis suggests a partial mixed-type inhibition mechanism. Both the Ki value of the inhibitor and the breakdown rate of the enzyme-substrate-inhibitor complex are dependent on the chain length of the PolyP, thus suggesting that the breakdown rate of the enzyme-substrate-inhibitor complex is regulated by the binding of Polyphosphate to a specific inhibitory site (Yoshino and Murakami, 1988). More complicated interactions were observed between PolyP and two oxidases, i.e. spermidine oxidase of soybeen seedling and bovine serum amine oxidase. PolyP competitively inhibits the activities of both enzymes, but may serve as an regulator because the amino oxydases are also active with the polyamine-PolyP complexes (Di Paolo et al., 1995). 

Two cytotoxic spermidine derivatives were isolated as a mixture from the Pacific soft coral Sinularia brongersmai 5,12-dimethyl-1-dimethylamino-5,9-diazaheneicos-l l-en-10-one (35) and its 11,12-dihydroderivative 36 (ratio 9 1). By hydrogenation of the mixture, a single compound was obtained that corresponded by GC/MS analysis to the minor component of the original mixture. Hydrolysis followed by esterification of the hydrogenated product 36 afforded methyl 3-methyldodecanoate and N,N,N-tri-methylspermidine. The results of a Hofmann degradation of 36 confirmed the conclusion on mass spectral evidence that the primary amide nitrogen is bonded to the trimethylene chain of the unsymmetrical spermidine molecule (61). 

Some alkaloids were found in nature to be derivatives of the celacinnine alkaloids, having an additional bond between the N1 substituent and the spermidine moiety. It seems that the precursors of the four alkaloids cyclo-celabenzine (134), isocyclocelabenzine (135), hydroxyisocyclocelabenzine (136), and pleurostyline (137) (Scheme 25) contain enamine systems that are able to cyclize. The structure elucidation of 134,135,136, and 137 was based on extensive spectroscopic analysis, mainly H- and 13C-NMR spectra of the alkaloids, their acetyl, and, in case of 137, their dihydro derivatives... 

The key alkaloid of the dihydroperiphylline group is periphylline (138), on which intensive chemical and spectroscopic analysis was performed. Essential to the structure determination was the detection of spermidine in an alkali melt of tetrahydroperiphylline (139), prepared by catalytic hydrogenation of periphylline (138). Alkali hydrolysis of 138 yielded ( -cin-... 

Verbascenine (205) has been isolated from the aerial parts of Verbascum species (140). Its structure elucidation was based on chemical degradation reactions as well as careful analysis of spectral data (140). In addition to spermidine, spermine was isolated from a KOH melt reaction and identified as its tetraacetyl derivative. The two cinnamic acid residues were identified by spectroscopic methods. A C=C double bond was found in only one of these units. The other cinnamic acid residue is part of a / -aminodihydrocin-namide. Therefore, verbascenine must be cyclic. The number of possible structures was reduced to two (205 and 206) on the basis of the formation of certain derivatives and their spectroscopic properties. Structure 205 is preferred for verbascenine because of the mass spectral behavior of its... 

The assay was carried out in phosphate buffer with radioactive putrescine, decarboxylated S-adenosylmethionine, and enzyme. Reactions were incubated at 37°C for 90 minutes and terminated by addition of perchloric acid. The solutions were clarified by centrifugation, and the polyamines were benzoyl-ated and extracted and then analyzed. Figure 9.55 shows the analysis of samples removed at zero time (blank) and in after 60 minutes incubation (sample) at 37°C. The appearance of radioactive spermidine is shown. The rate of product formation is shown in Figure 9.56. 

Analytical data C, 11.40% H, 2.20% N, 17.16% P, 25.50% and Cl, 43.45%. They are consistent with the crude structure N Cl (spermidine minus 3 H) C15P3N3 (mol. wt. = 731.186). In other words, elemental analysis would confer to the final product a SPIROBINO configuration in which the (NH2, NH) couple in H2N— (CH2)3— NH—(CH2)4—NH2 would link one N3P3C14 moiety in a SPIRO configuration when the last NH2 group would link one N3P3C15 moiety. 

A topologically novel bifunctional DNA intercalator (152) has been synthesised which is derived from the bisintercalator spermidine bisacridine (153). Studies conducted using uv spectrophotometry and viscometric analysis demonstrate that (153) is able to intercalate calf-thymus DNA. Modelling studies suggest that the best fit between DNA and (153) involves a catanated complex in which the long-axis of the acridine moieties and the base pairs are aligned in the normal manner. The formation of this unusual complex would require two base pairs to open. 

Recently, a LC/ESI-MS method for analysis of tyramine, trypt-amine, 2-phenylethylamine, histamine, cadaverine, putrescine, spermidine, and spermine in wine without any sample pretreatment, was... 

TABLE 8.3. Elution Program for LC/ESI-MS Analysis of Tyramine, Tryptamine, 2-Phenylethylamine, Histamine, Cadaverine, Pntrescine, Spermidine, and Spermine in Wine"... 

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