Application of colloidal palladium nanoparticles for labeling in electron microscopy

The application of palladium nanoparticles as electron-dense markers for labeling in both transmission and scanning electron microscopy requires their conjugation to a specific protein. The conjugation protocol described here includes the dihydrolipoic acid (DHLA) capping of Pd nanoparticles (8 nm equivalent diameter) and their subsequent covalent attachment to functional protein molecules such as streptavidin, protein A, or avidin. The single-step reaction was mediated using the cross-linking agent ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The final Pd conjugates were fully functional, as demonstrated by labeling of ultrathin resin sections of either bovine serum albumin or secretory granules of the salivary gland isolated from the partially fed female Ixodes ricinus tick. The results of bovine serum labeling were quantified, statistically evaluated, and compared with results obtained using commercially available gold particle conjugates (10 nm diameter). The highest values of labeling density were achieved using both streptavidin-Pd (106 ± 7 particles/μm2) and protein A-Au conjugates (130 ± 18 particles/μm2) compared to a commercial streptavidin-Au (66 ± 16 particles/μm2) and protein A-Pd conjugates (70 ± 11 particles/μm2). The concentrations of both DHLA and EDC, pH during conjugation, and finally thorough washing away of unbound proteins crucially influenced conjugation.     

Simultaneous determination of polyamines and catecholamines in PC-12 tumor cell extracts by capillary electrophoresis with laser-induced fluorescence detection

A method to detect polyamines and catecholamines in PC-12 tumor cell extracts by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) is described for the first time. Both derivatization conditions and buffer concentrations and pH were optimized. Under optimized conditions the polyamines (putresine, spermine, spermidine) and catecholamines (dopamine, norepinephrine, epinephrine, serotonin) were derivatized with fluorescein isothiocyanate and separated at 25 kV in a fused-silica capillary (50 microm ID x 40 cm) with 0.1 M borate, pH 9.0, in less than 18 min. The influence of running buffer conditions, such as buffer pH and concentrations, were also investigated. Linearity of the analytes ranged from 0.05 to 1.0 micromol/L, and the detection limit (S/N = 3 ) ranged from 0.03 to 2.50 nmol/L. The concentrations of polyamines and catecholamines in PC-12 tumor cell extracts were determined with this method.     

Oil in water emulsions stabilized by maillard conjugates of sodium caseinate-locust bean gum

Conjugates composed by sodium caseinate and locust bean gum (LBG) were produced through Maillard reaction in order to enhance their emulsifying properties. The reactions were performed (80?°C/76% relative humidity) at three protein/polysaccharide ratios (0.3, 0.6, 1.2), and the conjugates were added in emulsions at two pH values (3.5 and 7). The formation of protein-polysaccharide conjugates was confirmed by SDS-polyacrylamide gel electrophoresis and by FTIR spectra. At both pH values, the conjugates subjected to longer reaction times presented less emulsifying capability, probably due to insoluble compounds formation. Furthermore, the conjugates provided high stability in O/W emulsions at pH 7 due to the electrostatic repulsion of protein charged groups. However, even though the conjugates were not charged at pH 3.5, the stabilization process occurred at this condition probably due to the formation of a thicker interfacial layer, which provided an increase in steric repulsion between the droplets. Thus, the conjugates formed by sodium caseinate and locust bean gum can be characterized as a good emulsifying agent to be applied in different pH conditions.     

Light Scattering Study of the Antibody–Poly(methacrylic acid) and Antibody–Poly(acrylic acid) Conjugates in Aqueous Solutions

The effect of the conformational state of the polymer coil on the properties of protein–polymer conjugates has been studied for the conjugates of antibody (monoclonal antibody from 6C5 clone against inactivated rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase; Ab) with poly(methacrylic acid) (PMAA) or poly‐(acrylic acid) (PAA). The pH‐dependencies of molecular properties and structural parameters of aqueous solutions (radius of gyration, intensity of scattered light, hydrodynamic diameter, and polydispersity index) of Ab, PMAA, and PAA and their conjugates, i. e., Ab‐PMAA and Ab‐PAA, have been studied using static and dynamic light scattering techniques. While free Ab aggregates in solution and precipitates at its isoelectric point, the covalent attachment of a charged polymer to Ab prevents its association and shifts the precipitation point towards more acidic values (from pH 5.95 for Ab to pH ˜ 4.8 for Ab‐PMAA). The predominant role of the conformational status of the polymer in the process of conjugate precipitation has been considered. Contrary to the precipitation of Ab‐PMAA, the formation of stable colloidal particles was suggested for Ab‐PAA at pH < 4.8. In the conjugates, polymer chains surround the protein globule in an extremely compact manner while Ab significantly affects the polymer conformation. The essentially larger hydrodynamic radii of conjugates, when compared with their radii of gyration, confirm the strong interaction of conjugates with solvent molecules.     

In situ synthesis of gold and silver nanoparticles by using redox-active amphiphiles and their phase transfer to organic solvents

An in situ reduction approach to synthesizing gold and silver nanoparticles by using a series of newly designed, redox-active amphiphiles at basic pH is described. These amphiphiles are the conjugates of a fatty acid (e.g., oleic acid, stearic acid, and lauric acid) and a redox-active amino acid (e.g., tryptophan or tyrosine). The amphiphile-coated nanoparticles are then efficiently transferred from water to different nonpolar organic media (such as benzene, toluene, xylene, cyclohexane, and hexane) simply by acid treatment. The phase-transfer process was monitored by UV/visible spectroscopy and transmission electron microscopy, and the results showed that the average particle size and size distribution remain almost unchanged after transferring to the organic media. The anchoring of the amphiphile to the nanoparticle surface was confirmed by FTIR spectroscopy and thermogravimetric analysis. A mechanism is proposed to describe the stability of colloidal Au and Ag nanoparticles formed in situ and their phase transfer to organic solvents. The presence of the amphiphile increases the thermal stability of the colloidal gold nanoparticle conjugates in organic solvents.     

Itaconic acid carrier ampholytes for isoelectric focusing.

Commercial carrier ampholytes, obtained by coupling polyethylene polyamines to acrylic acid, exhibit a conductivity minimum in the pH range 5.5-6.5 owing to the lack of appropriate pK values of the polyamine in this pH region. By replacing acrylic with itaconic acid, it has been possible to effect substantial improvements in the pH range 5.5-6.5 as itaconic acid has a pK2 value of 5.45. Upon coupling, the pK of the gramma-carboxyl group remains virtually unaltered. With itoconic acid carrier ampholytes it has been possible to improve the conductivity in the pH range 5.5-6.5 by as much as 400% compared with conventional carrier ampholytes. It is suggected that the commercial products should be supplemented with itaconic acid carrier ampholytes in order to obtain a more uniform conductivity and buffering capacity in the pH range 3-10.     

The influence of synthetic conditions on the stability of methotrexate-monoclonal antibody conjugates determined by reversed phase high performance liquid chromatography.

Methotrexate (MTX) has been convalently attached to an IgG-type monoclonal antibody (791T/36) directed to tumour-associated antigen gp72. Conjugates were synthesized by the active ester method using MTX N-succinimidyl ester at various pH values (7.5-10.5). Following purification by gel filtration, high performance liquid chromatography was used to assess the free drug or its derivatives in samples of MTX-791T/36 conjugates previously treated (or not) with hydroxylamine. Quantitative analysis, performed on a reversed phase column (pore size 300 A) with isocratic acetonitrile-sodium acetate buffer (pH 4.8) as mobile phase, indicated no detectable amount of free methotrexate in hydroxylamine-treated conjugates even six months after their preparation. Similar observations were made with conjugates, whose synthesis were performed at pH greater than or equal to 10. In contrast, the presence of increasing amounts of drug/metabolite could be demonstrated in samples produced at lower pH values. Based on these findings, the pH-dependent kinetics of MTX release has been determined and used to design conditions under which stable MTX-791T/36 conjugates could be prepared without hydroxylamine reaction.     

Preparation and characterization of conjugates of silk fibroin and chitooligosaccharides

With the aim of the functionalization of silk fibroin (SF), conjugates of SF and polycationic chitooligosaccharides (COS) were prepared by the chemical modification of SF with cyanuric chloride (CY)-activated COS (COS-CY). The ¹H NMR spectrum of the reaction product between a model compound D-glucosamine and CY suggested that the COS-CY modifier was synthesized by the reaction of the amino group and the terminal anomeric hydroxyl group in COS, with the chlorine atom of CY. The ¹H NMR spectrum and amino acid analysis of the conjugates (COS-CY-SF) clarified that the tyrosine and lysine residues of SF reacted with a second chlorine atom of the triazine ring of the modifier. On the basis of the results of the hexosamine determination and the amino acid analysis of COS-CY-SF, it is estimated that COS-CY-SF consists of 38 wt% COS, 8 wt% CY, and 54 wt% SF. The absorbance at 600 nm as a function of pH for COS-CY-SF and SF indicated that the introduction of a large amount of hexosamine made SF amphiphilic and more water-soluble at lower pH values. The COS-CY-SF conjugates retarded the growth of Escherichia coli after incubation for 24 h at a conjugate concentration of 0.6% (w/v), while SF did not retard the growth at a SF concentration of 0.7% (w/v).     

The polyamines of Xanthium strumarium and their response to photoperiod

Flowering plants of Xanthium strumarium L., grown in 8 h photoperiods, were analysed for polyamines. Putrescine, spermidine and spermine were found throughout the plant in three forms: (a) as free polyamines; (b) conjugates soluble in 5% trichloracetic acid (TCA); and (c) bound to the TCA-insoluble precipitate. On a fresh weight basis, total polyamines are most abundant in young leaves and buds, especially flower buds. Spermidine predominates in the free polyamine fractions, while spermine is dominant in the conjugated fraction. Transfer of vegetative plants from 16 h photoperiods to 1, 2, 3, or 4 inductive cycles (8 h light + 16 h uninterrupted dark) caused rapid and marked changes in the polyamine titer of the leaves and ultimately, floral initiation. The titer of free putrescine per mg protein declined progressively with induction in all leaf sizes, while the titers of free spermidine and spermine rose during days 2 and 3 in small and expanding leaves. Conjugated putrescine, spermidine and spermine rose sharply after only 1 inductive cycle, especially in small and expanding leaves, and maintained the higher level for at least several cycles. In plants given 4 inductive cycles, buds harvested after 4 additional days had sharply elevated levels of conjugated polyamines, especially spermine, on a protein basis.     

A radioimmuno-chromatographic scanning method for the analysis of testosterone conjugates in urine and serum

A method for the analysis of testosterone (and 5alpha-dihydrotestosterone) conjugates in human serum and urine samples is described. The samples were grought to pH 1 and extracted with a diethyl ether-methanol mixture. After evaporation the residues were run in a thin-layer chromatography system, individual samples' paths were cut into 1-cm long pieces and eluted with methanol. The methanol was evaporated and the residue subjected to acid hydrolysis. The released steroid was extracted by diethyl ether and measured by radioimmunoassay. The methodology described represents a new approach to the qualitative and quantitative study of steroid conjugates in serum and urine, and can easily be applied to the study of steroid conjugates in other biological mateiral.     

胺类分子结构对油酸囊泡pH窗口的影响

基于激光丁达尔效应及浊度测定, 考察了改变二元胺的碳桥长度、 多元胺的氨基多寡、 长链伯胺的碳链长度及季铵化等因素对油酸囊泡pH窗口的影响. 结果表明, 二元胺及多元胺主要导致油酸囊泡的pH窗口向碱性方向拓宽, 而长链伯胺和十二烷基三甲基溴化铵(DTAB)不仅可使油酸囊泡的pH窗口向碱性拓宽, 而且可使油酸在酸性pH区域形成另一个介稳至稳定的新囊泡相. 氨基在不同pH下质子化和脱质子化转换是胺类分子调节油酸囊泡pH窗口的共同驱动力, 疏水作用是长链胺类的又一驱动力, 而静电吸引是季铵盐的另一种特殊驱动力. 分子间相互作用的热力学参数及结合能计算结果表明, 二乙烯三胺为代表的二元胺或多元胺在油酸囊泡表面以氢键或离子-偶极作用等非共价结合为主, 其调节功能弱于长链伯胺及DTAB与油酸的疏水共组装或DTAB与油酸的静电吸引作用.     

Mass fragmentographic identification of polyamine metabolites in the urine of normal persons and cancer patients, and its relevance to the use of polyamines as tumour markers

The mass fragmentographic identification of N-(2-carboxyethyl)-4-amino-n-butyric acid, N-(3-aminopropyl)-N1-(2-carboxyethyl)-1,4-diaminobutane, N,N1-bis(2-carboxyethyl)-1,4-diaminobutane, and delta-aminovaleric acid in acid-hydrolysed urines of a normal person and two cancer patients is described. A previous study, in which the metabolic fate of intraperitoneally injected polyamines in rats was investigated, revealed that these compounds should be considered as non-alpha-amino acid metabolites of the naturally occurring polyamines. Quantification of polyamines and their non-alpha-amino acid metabolites by gas chromatography with nitrogen--phosphorus detection showed that, relative to the parent polyamines, humans normally excrete higher quantities of polyamine catabolites in urine than rats, suggesting that humans catabolize polyamines more efficiently. As illustrated by the follow-up of the concentrations of polyamines and their catabolites in the urine of a patient with high-grade non-Hodgkin lymphoma during chemotherapy, the catabolic pressure on polyamines may be considerably increased during neoplastic diseases, since an even higher proportion of oxidized polyamine metabolites was observed. It is therefore suggested that the additional measurement of the circulating concentrations of polyamine-degrading enzymes is of importance for the correct interpretation of polyamine (metabolite) determinations for oncological purposes.     

肿瘤酸度响应性聚(L-赖氨酸)-阿霉素键合药的制备与表征

用丁二酸酐(SA)和顺式乌头酸酐(CA)分别对阿霉素(DOX)进行修饰, 得到非酸响应的SA-DOX(SAD)和酸响应的CA-DOX(CAD). 通过SAD或CAD、端羧基化的聚乙二醇单甲醚(mPEG-COOH)与聚(L-赖氨酸)(PLL)的缩合反应, 制得非酸响应的PLL-g-mPEG/SAD和酸响应的PLL-g-mPEG/CAD键合药. 通过核磁共振氢谱和红外光谱表征键合药的化学结构, 并通过紫外-可见分光光度计测定药物键合量. 动态激光光散射研究结果表明, 两亲性的PLL-DOX键合药可以在pH=7.4的磷酸缓冲溶液中自组装形成稳定的纳米微粒. 体外释放实验及噻唑蓝检测结果表明, PLL-g-mPEG/SAD在实验pH范围和时间段内只释放出少量DOX, 不具有酸响应特性, 且对HeLa细胞增殖抑制作用较小. 而PLL-g-mPEG/CAD在生理条件(pH=7.4)下相对稳定, 在弱酸性条件(pH=5.3, 6.8)下, CAD中酸响应的酰胺键能快速水解并释放出DOX, 表现出较强的HeLa细胞增殖抑制效果.     

Selective fluorometric detection of polyamines using micellar electrokinetic chromatography with laser-induced fluorescence detection

The polyamines putrescine, cadaverine, spermine and spermidine were separated and quantified by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. The derivatization reagent, 1-pyrenebutanoic acid succinimidyl ester (PSE), allowed for the selective detection of the polyamines at 490 nm. Multiple labeling of the polyamines with PSE allows the formation of intramolecular excimers that emit at longer wavelengths (450-520 nm) than mono-labeled analytes (360-420 nm). Optimal separation of the labeled polyamines was achieved using a separation buffer consisting of 10 mM phosphate pH 7.2, 30 mM cholate, and 30% acetonitrile. Using these conditions, the four polyamines were separated in under 10 min. Limits of detection for putrescine, cadaverine, spermine and spermidine were 6, 5, 15 and 13 nM, respectively. These are superior or comparable to those previously reported in the literature using fluorescence detection.     

Recent Advances in the Synthesis of Polyamine Derivatives and Their Applications

Biogenic polyamines (PAs) are involved in the growth and development of normal cells, and their intracellular concentration is stable. The concentration of PAs in cancer cells is significantly increased to promote and sustain their rapid proliferation. Over the years, synthetic PAs, which differ in their structure, have demonstrated high antitumor activity and are involved in clinical trials. The chemical synthesis of PAs and their conjugates require the correct choice of synthetic pathways—methods for constructing conjugates and the orthogonal protection of amino groups. The most common methods of synthesis of PA conjugates are acylation of regioselectively protected PAs or their alkylation under the conditions of the Fukuyama reaction. One of the most promising methods of PA synthesis is the use of a multicomponent Ugi reaction, which allows various PAs to be obtained in high yields. In this review, we describe and analyze various approaches that are used in the synthesis of polyamines and their conjugates.     

Reversed-phase ion-pair liquid chromatographic procedure for the simultaneous analysis of S-adenosylmethionine, its metabolites and the natural polyamines

A method using reversed-phase ion-pair liquid chromatography with dual detection was developed for the simultaneous determination of the S-adenosylmethionine (SAM) analogues and the natural polyamines. The separation is obtained with a gradient elution and by adjusting the concentration of octanesulfonic acid used as ion-pairing agent, the ionic strength of the eluent, the pH and the acetonitrile content of the eluents. The SAM analogues are analyzed by UV detection at 254 nm and the polyamines by fluorescence detection after post-column derivatization with o-phthalaldehyde. The method allows the determination of the SAM analogues and the polyamines in one single run by direct injection of tissue extracts. The procedure is applied to the study in rats and in hepatoma tissue culture cells of the biochemical effects of alpha difluoromethylornithine, a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase.     

Micellar electrokinetic chromatography of polyamines and monoacetylpolyamines

A selective procedure for qualitative and quantitative analysis of ten polyamines by micellar electrokinetic chromatography (MEKC) was developed. Benzoylated polyamines and acetylpolyamines in micellar phase of SDS (10 mM) were separated at 25 degrees C by 20 mM borate buffer pH 8.5, containing 8% ethanol, with an applied voltage of 25 kV (5 microA) and then detected at 198 nm. The experimental factors and operational parameters were optimized by performing analysis at different surfactant concentrations, pH, voltage and temperature with and without ethanol. The repeatibility of migration times and peak heights is a peculiarity of the method here described.     

High-performance liquid chromatography of glutathione conjugates

Summary An ion-exchange high-performance liquid chromatographic method is described for the quantitative assay of glutathione (GSH) conjugates derived from endogenous electrophilic substances as well as xenobiotics. GSH (reduced and oxidized forms) and GSH conjugates were condensated with o-phthaldialdehyde to highly fluorescent derivatives and monitored at 338 nm excitation and 450 nm emission wavelengths after separation by ion-exchange HPLC on a 60-5NH₂ Polygosil analytical column. The detection limit was 2 pmol for the GSH conjugate of cholesterol epoxide and 6 pmol for the GSH conjugate of oleic acid epoxide. This method allows sensitive determination of all GSH conjugates independent of the chromatographic and spectrophotometric properties of the electrophilic substrates. Using this method we could show for the first time that the endogenous compound oleic acid epoxide is a specific substrate for the cytosolic rat liver GSH S-transferase. The method is applied to the determination of GSH S-transferase activity towards oleic acid epoxide and cholesterol epoxide.     

Evaluation of conditional stability constants of dissolved aluminum/humic substance complexes by means of 27Al nuclear magnetic resonance

An analytical procedure for evaluating the conditional stability constants K′ of aluminum/humic substance complexes, based on ²⁷Al nuclear magnetic resonance spectroscopy, is described. It is well suited for the determination of Al(III) dissociated from dissolved humic substances at slightly acidic pH values. The new procedure offers a good sensitivity and requires neither special preparation of the sample nor the evaluation of complex coupled chemical equilibria. The pK′ values range from 2.0 to 4.3 depending on the ratio [Al_complex] to dissolved organic carbon and the pH.     

Synthesis of HPMA Copolymers Containing Doxorubicin Bound via a Hydrazone Linkage. Effect of Spacer on Drug Release and in vitro Cytotoxicity

The aim of this study was the synthesis, physico‐chemical characterization and preliminary evaluation of biological activity of novel polymer drugs based on conjugates of anti‐cancer drug doxorubicin (Dox) with water‐soluble polymer drug carriers based on N‐(2‐hydroxypropyl)methacrylamide (HPMA) copolymers. In the conjugates, Dox is attached to the polymer via a pH‐sensitive linkage susceptible to hydrolysis at pH ≈ 5–6, thus enabling intracellular Dox release. Seven Dox‐containing polymer conjugates differing in the length and structure of the single‐amino‐acid or oligopeptide spacer were synthesized (Gly, β‐Ala, 6‐aminohexanoyl, 4‐aminobenzoyl, GlyGly, GlyLeuGly, Gly‐DL ‐PheLeuGly) and the relationship between the spacer structure and the rate of in vitro Dox release was studied at various pH. The rate of Dox release at pH 5 (close to lysosomal pH) ranged from 70 to 96% of total Dox/48 h, depending on the spacer structure and being the highest for the conjugate containing the 6‐aminohexanoyl spacer. The rate of Dox release from most conjugates incubated at pH 7.4 (blood pH) was more than 10 times slower, ca. 4–10% of total Dox/48 h. Molecular weight of the polymer (25 000–115 000 g/mol) did not significantly influence the rate. The presence of lysosomal enzyme cathepsin B in incubation media increased the rate of Dox release from the conjugates with oligopeptide GlyLeuGly and Gly‐DL ‐PheLeuGly spacers by 15–30%, whereas the release from conjugates with other spacers remained unchanged. Cytotoxicity of all hydrazone conjugates for mouse EL‐4 T cell lymphoma cells was much higher and close to that of free Dox (IC₅₀ ≈ 0.1–0.34 μg Dox/mL), in contrast to cytotoxicity of similar classic conjugates bearing Dox attached via an amide bond (IC₅₀ ≈ 19 μg Dox/mL).