Gel Electrophoretic Studies Research Articles

A Spectroscopic Approach to Unravel the Local Conformations of a G-Quadruplex Using CD-Active Fluorescent Base Analogues.

The formation of a stable G-quadruplex (GQ) can inhibit the increased telomerase activity that is common in most cancers. The global structure and the thermal stability of the GQs are usually evaluated by spectroscopic methods and thermal denaturation properties. However, most biochemical processes involving GQs might require local conformational changes at the guanine tetrad (G4) level. These local conformational changes of individual G4 layers during protein and drug interactions have not yet been explored in detail. In this study, we monitored the local conformations of individual G4 layers in GQs using 6-methylisoxanthopterine (6MI) chromophores, which are circular dichroism (CD)-active fluorescent base analogues of guanine, as local conformational probes. A synthetic, tetramolecular, parallel GQ with site-specifically positioned 6MI monomers or dimers was used as the experimental construct. Analytical ultracentrifugation studies and gel electrophoretic studies showed that properly positioned 6MI monomers and dimers could form stable GQs with CD-active fluorescent G4 layers. The local conformation of individual fluorescent G4 layers in the GQ structure was then tracked by monitoring the absorbance, fluorescence intensity, thermal melting, fluorescence quenching, and CD changes of the incorporated probes. Overall, these studies showed that site-specifically incorporated fluorescent base analogues could be used as probes to monitor the local conformational changes of individual G4 layers of a GQ structure. This method can be applied to explore the details of small molecule-GQ interaction at the level of the individual G4 layers, which may prove to be useful in designing drugs to treat GQ-related genetic disorders, cancer, and aging.

Influence of the extent of enzymatic hydrolysis on the functional properties of protein hydrolysates from visceral waste of Labeo rohita

The study aimed to evaluate the extent of hydrolysis on the functional properties of fish protein hydrolysates from fish wastes. Fish protein hydrolysate (FPH) was prepared from the visceral waste of Rohu (Labeo rohita) using Alcalase®. Independent variables of the hydrolysis conditions, namely, time, temperature and enzyme-substrate ratio were optimized through response surface methodology using a completely randomized factorial design. Functional properties of FPH, such as antioxidant activity, metal chelating activity, emulsifying and foaming capacity were studied. Pre-heating of the raw viscera resulted 83% reduction of lipid content in the FPH. The DH was found to be significantly influenced by the hydrolysis conditions studied. The gel electrophoretic study showed that FPH contained peptides in the range of < 10-25kDa. Optimum degree of hydrolysis (DH) was obtained as 34.7% at 87.5min, 48.64°C and enzyme concentration of 0.99%. The results demonstrated that the antioxidant and metal chelating properties of FPH increased with the increase of DH. The surface active properties like emulsifying and foaming capacity were found to be decreased with the increased levels of hydrolysis. These results suggest that hydrolysates from visceral waste of Rohu could find potential use as supplement in animal feed.

Development of Functional Nanomaterials from Wheat Bran Derived Arabinoxylan for Nucleic Acid Delivery.

Wheat bran is a major byproduct of the wheat industry and a rich source of cellulosic and hemicellulosic compounds. We developed a facile and reproducible method to generate functional nanomaterials from wheat bran derived polysaccharide, Arabinoxylan (AX). We first established that AX derived from wheat bran was chemically equivalent to commercially available AX extracted from wheat flour. Through facile chemical modification, positive and negatively charged domains were introduced along AX backbone, which in turn induced local electrostatic and hydrophobic interactions promoting the formation of nanoparticulate structures. The extracted, chemically modified AX was characterized using FTIR, 1H NMR, and elemental analysis. We observed that, while both anionic and cationic AX self-assemble into stable, spherical nanoparticles with a low polydispersity index, the unmodified AX did not exhibit such self-organizational properties. To form functionally active nanomaterials, we further complexed negatively charged CRISPR-Cas9 DNA with cationic AX. Through gel electrophoretic studies, we identified that, at a feed ratio of DNA to AX of 1:15, AX is capable of forming polyplexes with DNA in the form of nanoparticles with an average hydrodynamic diameter of ∼100 nm and surface charge of -1.40 ± 0.91 mV. We envision that chemically modified AX, originally sourced from agricultural waste materials and not from food products, can be used as functional nanomaterials for gene delivery in the agrochemical sector thus catalyzing the circular approach of sustainability.

Radiation induced DNA damage and its protection by a gadolinium(III) complex: Spectroscopic, molecular docking and gel electrophoretic studies.

The current work describes the efficacy of an artificially synthesized Gd(III) complex as a potential radioprotecting molecule. The work involves utilization of spectroscopic and electrophoretic techniques to investigate the radioprotecting behavior of the Gd(III) complex. Spectroscopic studies revealed that the complex interacted strongly with DNA while molecular docking studies suggested groove binding through H-bond formation and other non-covalent interactions. The Gd(III) complex was found to impart 94% and 91% protection to irradiatively damaged DNA at radiation doses of 20 and 25 Gy respectively. The protection is believed to occur via radical scavenging mechanism and the antioxidant behavior of the complex suggested a strong radical scavenging property.

Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae.

Molecular combing and gel electrophoretic studies revealed endogenous nicks with free 3′OH ends at ∼100 kb intervals in the genomic DNA (gDNA) of unperturbed and G1-synchronized Saccharomyces cerevisiae cells. Analysis of the distribution of endogenous nicks by Nick ChIP-chip indicated that these breaks accumulated at active RNA polymerase II (RNAP II) promoters, reminiscent of the promoter-proximal transient DNA breaks of higher eukaryotes. Similar periodicity of endogenous nicks was found within the ribosomal rDNA cluster, involving every ∼10th of the tandemly repeated 9.1 kb units of identical sequence. Nicks were mapped by Southern blotting to a few narrow regions within the affected units. Three of them were overlapping the RNAP II promoters, while the ARS-containing IGS2 region was spared of nicks. By using a highly sensitive reverse-Southwestern blot method to map free DNA ends with 3′OH, nicks were shown to be distinct from other known rDNA breaks and linked to the regulation of rDNA silencing. Nicks in rDNA and the rest of the genome were typically found at the ends of combed DNA molecules, occasionally together with R-loops, comprising a major pool of vulnerable sites that are connected with transcriptional regulation.

Platinum(II) Iodido Complexes of 7-Azaindoles with Significant Antiproliferative Effects: An Old Story Revisited with Unexpected Outcomes.

A series of platinum(II) diiodido complexes containing 7-azaindole derivatives, having the general formula cis-[PtI2(naza)2] (1–8), has been prepared and thoroughly characterized, including X-ray structure analysis of cis-[PtI2(2Me4Claza)2]∙DMF (8∙DMF; 2Me4Claza = 2-methyl-4-chloro-7-azaindole). Complexes showed high in vitro cytotoxicity against nine human cancer cell lines (IC50 ranging from 0.4 to 12.8 μM), including the cisplatin-resistant ovarian cancer cell line (A2780R; IC50 = 1.0–3.5 μM). The results of in vivo testing, using the L1210 lymphocytic leukaemia model, at the equimolar doses of Pt with cisplatin (2 mg/kg) confirmed the activity of complex 8 comparable to cisplatin. From the mechanistic point of view, evaluated ex vivo by Western blot analyses on the samples of isolated tumour tissues, the treatment of the animals with complex 8, contrary to cisplatin, decreased the levels of tumour suppressor p53 and increased significantly the amount of intracellular anti-apoptotic protein MCL-1L (37 kDa). Additionally, the active form of caspase 3 was significantly elevated in the sample of tumour tissues treated with complex 8, indicating that the activation of p53-independent cell-death pathway was initiated. The light and electron microscopy observations of the cancerous tissues revealed necrosis as a dominant mechanism of cell death, followed by scarce signs of apoptosis. The additional results (e.g. in vitro interaction experiments with selected biomolecules, cell cycle perturbations, gel electrophoretic studies on pUC19 plasmid DNA) supported the hypothesis that the complexes might be involved in the mechanism of action quite different from cisplatin.

Chemical and microbial properties of shidal, a traditional fermented fish of Northeast India.

Like Southeast Asian countries fermented fish is a well known strategy of food preservation in the Northeast region of India. Shidals are mostly preferred salt-free fermented fish product amongst all. Chemical and microbial composition, antioxidative potential, fatty acid profile and proteins profile in gel electrophoresis of shidals were studied. pH and total titratable acidity (TTA) have been found as 5.86±0.11, 0.115±0.01 and 6.62±0.07, 0.092±0.01 in punti and phasa shidal respectively. DPPH (·) radical scavenging activity of punti and phasa shidal was determined as 80.15±5.67 and 68.30±3.22 respectively. Presence of eicosapentaenoic, docosahexaenoic, arachidonic, linolenic and linoleic acid indicate the nutritional significance of shidal. However, the result showed that punti shidal was rich in omega-3 but poor in omega-6 fatty acid, whereas, reverse was observed in respect of phasa shidal. Poly acrylamide gel electrophoretic study of protein revealed disappearance of myosin head chain (MHC) in the dry puntius fish (raw material of shidal). Proteins or peptides with low molecular weight between 45 and 29kDa and between 45 and 66kDa were noticed in both the shidals and indicative of intensive protein degradation during fermentation. Therefore, fermented fish product, shidal could be used as a potential source of nutrients and natural antioxidants.

Ruthenium(II)-arene complexes with naphthalimide-tagged N,O- and N,N-chelating ligands: Synthesis and biological evaluation

A new family of ruthenium(II)-arene complexes with naphthalimide functionalized N,O- and N,N-chelating ligands of the general formula [Ru(η6-p-cymene)Cl(L)] (2b–4b) (where: L=4-[N-(2-((2-hydroxy-5-Br-phenyl)methyl imino)ethyl)]-N-butyl-1,8-naphthalimide (2a), 4-[N-(2-((2-hydroxy-5-Cl-phenyl)methyl imino)ethyl)]-N-butyl-1,8-naphthalimide (3a), and N-butyl-4-[N-(2-((2-hydroxy-5-NO2-phenyl)methylimino)ethyl)]-N-butyl-1,8-naphthalimide (4a), and [Ru(η6-p-cymene) Cl(L′)]Cl (8b–9b) (where L′=N-(2,2′-dipyridylaminoethyl)-1,8-naphthalimide (8a) and N-(2,2′-dipyridylaminopropyl)-1,8-naphthalimide (9a) have been synthesized and characterized. The in vitro cytotoxic activities of the ligands (2a, 9a) and the complexes (2b–4b, and 8b–9b) have been evaluated against the human melanoma skin cancer (CRL7687) and normal noncancerous (CA-M75) cell lines. All the compounds exhibit potent cytotoxic activities with IC50 values of ∼1μM or less but displayed variable selectivity. The compounds with N,O-ligands were found to be less selective than those containing N,N-chelating ligands. Notably, complex 9b displayed the highest selectivity towards cancer cells over health cells. The interactions of the compounds with calf thymus DNA (CT-DNA) have also been investigated by UV–Vis and fluorescence spectra, ethidium bromide displacement assay and gel electrophoretic studies, which revealed that the compounds bind to CT-DNA moderately presumably through an intercalative mode.

Exploration of DNA binding mode, chemical nuclease, cytotoxic and apoptotic potentials of diketone based oxovanadium(IV) complexes

Two diketone based oxovanadium complexes, viz., bis(4,4,4-trifluoro-1-phenylbutane-1,3-dionato)oxovanadium(IV) (1) and bis(1,1,1-trifluoropentane-2,4-dionato)oxovanadium(IV) (2), have been synthesized and characterized by spectroscopic and analytical techniques. The DNA binding and the cleaving ability of the complexes is assessed by UV–vis spectroscopy, fluorescence spectroscopy, viscometry and gel electrophoretic studies. The DNA binding constant values (Kb) are found to be 1.95±0.16×103M−1 for complex 1 and 1.064±0.17×103M−1 for complex 2, respectively. Based on the results of the spectral and viscosity studies, it is observed that the complexes, interestingly, have preferred minor groove binding with DNA. Further, the concentration-dependent oxidative cleavage pattern of pBR322 in the presence of the activating reagent, hydrogen peroxide, has also been discussed. In addition, the complexes have shown moderate cytotoxic activity by inducing apoptosis against the cervical cancer cell line, HeLa. The results of in silico analysis and logP predictions are found to be in good agreement with the experimental observations. Thus, synthesized oxovanadium complexes have displayed promising DNA binding behavior and DNA cleavage activity with moderately cytotoxic nature.

SPION-mediated soil DNA extraction and comparative analysis with conventional and commercial kit-based protocol

Direct isolation of soil DNA comes as an emerging technology to understand the microbial diversity of a particular environment circumventing the dependency on culturable methods. Soil DNA isolation is tough due to the presence of various organic components present in soil which interfere in extraction procedure. Here, we report a novel direct soil DNA extraction protocol utilizing bare superparamagnetic iron oxide nanoparticles and its comparison with conventional and commercial kit-based soil DNA extraction methods. The quality, quantity and feasibility of the recovered DNA from all the three methods towards various molecular techniques were checked. Our magnetic nanoparticle-based soil DNA extraction successfully yields pure DNA without any RNA or protein contamination as revealed by the nanodrop spectrophotometer and agarose gel electrophoretic study. Different methods of soil DNA extraction were evaluated on the basis of PCR, denaturing gradient gel electrophoresis and real-time PCR. Soil DNA extracted using conventional method fails to carry out critical molecular biology techniques where as magnetic nanoparticle-based soil DNA extraction gave good results which is comparable to commercial kit. This comparative study suggests that protocol described in this report is novel, less time consuming, cost effective with fewer handling steps and yields high quantity, good quality DNA from soil.

Interaction between Neuronal Nitric-Oxide Synthase and Tetrahydrobiopterin Revisited: Studies on the Nature and Mechanism of Tight Pterin Binding

Recombinant neuronal nitric-oxide synthase (nNOS) expressed in baculovirus-infected Sf9 cells contains approximately 1 equiv of tightly bound tetrahydrobiopterin (BH4) per dimer and binds a second equivalent with a dissociation constant in the 10–7–10–6 M range. Less is known about the pterin-binding properties of nNOS originating from expression systems such as Escherichia coli that do not produce BH4. We determined the binding properties of E. coli-expressed nNOS for BH4 and several inhibitory pterins by monitoring their effects on enzyme activity. E. coli-expressed nNOS as isolated was activated by BH4 monophasically with EC50 ≈ 2 × 10–7 M, demonstrating a lack of tight pterin binding. However, overnight incubation with BH4 resulted in tight binding of one BH4 per dimer, yielding an enzyme that resembled Sf9-expressed nNOS. Tight pterin binding was also induced by preincubation with 4-amino-tetrahydrobiopterin, but not by 7,8-dihydrobiopterin or 4-amino-dihydrobiopterin, suggesting that tight-binding site formation requires preincubation with a fully reduced pteridine. Kinetic experiments showed that tight-binding site formation takes approximately 10 min with 1 μM BH4 (2 min with 1 μM 4-amino-BH4) at 4 °C. Anaerobic preincubation experiments demonstrated that O2 is not involved in the process. Gel electrophoretic studies suggest that tight-binding site formation is accompanied by an increase in the strength of the NOS dimer. We propose that incubation of pterin-free nNOS with BH4 creates one tight pterin-binding site per dimer, leaving the other site unaffected, in a reaction that involves redox chemistry.

Disulphide Linkages Occur in Many Polypeptides of Rice Protein Fractions: a Two-dimensional Gel Electrophoretic Study

Most of the work on rice storage proteins focused on the major fraction – glutelin. In contrast to previous reports of the occurrence of single legumin-like polypeptide pair in glutelin, two-dimensional gel electrophoresis revealed many glutelin bands, with some having and some lacking disulphide-linkages. Five legumin-like polypeptide pairs exhibited wide heterogeneity over a range of molecular weight ( M r ) 25 to 60 kDa, each having a large subunit ( M r ranged from 18 to 40.5 kDa) disulphide-bonded to a small subunit ( M r ranged from 16.5 to 18.0 kDa). A band of 49 kDa was homodimeric with two subunits of 29 kDa each; a polypeptide of 51 kDa which altered position to ones corresponding to 53 kDa and 57 kDa on 2-D gels contained intrapolypeptide linkages. Polypeptides of 65 kDa and 60 kDa occurred as aggregates of 110 kDa. The number of polypeptides in other seed protein fractions albumins, globulins and prolamins, varied from four to ten. One of the albumin bands had intrapolypeptide disulphide linkages (20 kDa) and the globulins contained two such bands (13.5 and 20 kDa). Thus, the present study provides a description of the polypeptide composition of different rice protein fractions that is finely resolved with respect to the occurrence of disulphide linkages.

Cytotoxic effect of benzo(a)pyrene on development and protein pattern of sunflower pollen grains

Benzo(a)pyrene (BaP) is an important part of diesel exhaust particles (DEP). The overall objective of this research was to elucidate some microscopic effects of BaP on the formation, development, and structure of pollen grains and its proteins in sunflower (Helianthus annuus L). Sunflower plants were grown in experimental pots and treated with the different concentrations of BaP. Flowers and young buds were removed, fixed in FAA70 (formalin:acetic acid:ethanol, 2 : 1 : 17) and subjected to developmental studies. Our results show that BaP-treatment causes abnormalities during pollen development. Shape of microspore tetrads changed from spherical to polygonal or irregular. Pollen grains in normal plants are spherical in both equatorial and polar views; in BaP-treated plants, they changed into irregular shapes in equatorial view and triangular shape in polar view. Delay in degeneration of the tapetum layer in anther, and formation of giant, irregular, and non-fertile pollen grains are other results of BaP treatment. Gel electrophoretic studies revealed that a band with a molecular mass of 30 kDa disappeared in the BaP-treated group and two new proteins with molecular masses of 15 and 20 kDa were formed. Some pollen proteins may act as allergens, considering the fact that pollen allergy frequency is increased in polluted areas, especially DEP-polluted ones; the possibility arises that BaP, as an important part of DEP, could be an effective agent entailing formation of detoxifying proteins which, on the other hand, can also act as allergens.

Effect of the water-soluble fraction of diesel exhaust particles on the development and protein patterns of pollen grains in bean plants (Phaseolus vulgaris L.)

Excessive air pollution may adversely affect plant growth and development. The overall objective of this research was to elucidate some microscopic effects of diesel exhaust particles (DEP) on the formation, development, and structure of pollen grains and its proteins in Phaseolus vulgaris L. Bean plants were grown in experimental pots and treated with different concentrations of the water-soluble fraction of DEP. Flowers and young buds were removed, fixed in FAA70 (formalin : acetic acid : ethanol, 2 : 1 : 17), and subjected to developmental studies. Our results show that DEP cause some abnormalities during pollen development. Pollen grains in normal plants are spherical in equatorial view and triangular in polar view; in DEP-treated plants, they were changed into irregular shapes in equatorial view and asteroidal in polar view. Delay in degeneration of the tapetum layer of the anther, and formation of giant, irregular, and non-fertile pollen grains are other results of DEP treatment. Gel electrophoretic studies revealed that a new protein with a molecular mass of 80 kD was formed in the DEP-treated group. Some pollen proteins may act as allergens, considering the fact that pollen allergy frequency is increased in polluted areas; the possibility arises that DEP could be an effective agent entail formation of detoxifying proteins which, on the other hand, can also act as allergens.

Improvement of Gelling Properties of Lizardfish Mince as Influenced by Microbial Transglutaminase and Fish Freshness

The effects of microbial transglutaminase (MTGase) at different levels (0 to 0.8 units/g sample) on the properties of gels from lizardfish (Saurida undosquamis) mince set at 25 degrees C for 2 h or 40 degrees C for 30 min prior to heating at 90 degrees C for 20 min were studied. Breaking force and deformation of gels increased with increasing MTGase amount added (P<0.05). At the same MTGase level used, gels with the prior setting at 40 degrees C for 30 min showed a higher breaking force compared with those subjected to prior setting at 25 degrees C for 2 h (P<0.05). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic study revealed that myosin heavy chain (MHC) underwent polymerization to a higher extent in the presence of MTGase. Regardless of setting condition, microstructure of gel added with MTGase was finer with a smaller void compared with that of gel without MTGase. Therefore, setting temperature affected the property of gels added with MTGase. Gel properties of mince obtained from lizardfish stored in ice for different times (0 to 10 d) with and without MTGase at a level 0.6 units/g were determined. Irrespective of MTGase addition, breaking force and deformation of all gels decreased as the storage time of lizardfish increased (P<0.05). The addition of MTGase was able to increase both breaking force and deformation of the resulting gel produced from lizardfish kept in ice for all storage times used. Therefore, both freshness and MTGase addition had the direct impact on gel properties of lizardfish mince.

Glycine Transporter Dimers: EVIDENCE FOR OCCURRENCE IN THE PLASMA MEMBRANE

Different Na(+)/Cl(-)-dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuT(Aa), as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs.

Reversed‐phase high‐performance liquid chromatographic, size exclusion chromatographic and polyacrylamide gel electrophoretic studies of glycinin: evidence for molecular species and their association–dissociation

Isolation and purification of glycinin and its molecular species from an Indian soybean variety (JS-335) was achieved using polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). Glycinin was found to have two molecular species (glycinin I and II), and only glycinin I underwent reversible dissociation-association system into alpha and beta species. Glycinin I and II were not found to constitute a dissociation-association system. Glycinin II also did not dissociate under varying conditions of time, pH and ionic strength of buffer. Various species so dissociated were isolated, purified and characterized.

Gelling properties of white shrimp ( Penaeus vannamei) meat as influenced by setting condition and microbial transglutaminase

The properties of white shrimp ( Penaeus vannamei) gel added with different levels of microbial transglutaminase (MTGase) and subjected to setting at 25 °C for 2 h or 40 °C for 30 min, prior to heating at 90 °C for 20 min were studied. Breaking force of gels with and without setting increased with increasing MTGase amount added ( P < 0.05 ). However, no changes in deformation in all samples were noticeable ( P > 0.05 ). Directly heated gels showed the lower breaking force than those with prior setting at all MTGase levels added ( P < 0.05 ). Generally, gels prepared by setting at 25 °C exhibited the greater breaking force than those set at 40 °C, possibly associated with the appropriate protein structure for cross-linking at 25 °C and greater degradation at 40 °C as evidenced by a greater trichloroacetic acid soluble peptide content ( P < 0.05 ). Sodium dodecyl sulfate polyacrylamide gel electrophoretic study revealed that myosin heavy chain (MHC) underwent polymerization to a higher extent in the presence of MTGase, but the strengthening effect on gel was dependent on setting temperature. Regardless of setting condition, microstructure of gel added with MTGase was finer with a smaller void, compared with those of gel without MTGase. Therefore, setting temperature played an essential role in gel property of white shrimp meat added with MTGase.

Crude Protein Electrophoresis Of Selected Species Of &lt;i&gt;Senna&lt;/i&gt; Miller

Polyacrylamide gel electrophoretic studies were conducted with seed protein extracts of four species of Senna and a simple-leafed accession. The observed protein bands revealed some measure of biogenetic relationship among the species, through insufficient as a diagnostic aid at infra-generic level. The bands, however, appear useful at the generic level and above. The sectional distinction achieved by the protein profile is noted. Key words: electrophoresis, Senna, systematics, seed protein Ife Journal of Science Vol. 7(1) 2005: 59-62

Enhancing Effects of Galactosylated Dendrimer/.ALPHA.-Cyclodextrin Conjugates on Gene Transfer Efficiency

To improve in vitro gene transfer efficiency and/or achieve cell-specific gene delivery of polyamidoamine (PAMAM) starburst dendrimer (generation 2, G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugate bearing galactose (Gal-alpha-CDE conjugates) with the various degrees of substitution of the galactose moiety (DSG) as a novel non-viral vector. The agarose gel electrophoretic studies revealed that Gal-alpha-CDE conjugates formed complexes with plasmid DNA (pDNA) and protected the degradation of pDNA by DNase I, but these effects impaired as the DSG value increased. Dendrimer and alpha-CDE conjugate exerted pDNA condensation through the complexation, but Gal-alpha-CDE conjugates did not. Gal-alpha-CDE conjugate (DSG 4) was found to have much higher gene transfer activity than dendrimer, alpha-CDE conjugate and Gal-alpha-CDE conjugates (DSG 8, 15) in HepG2, NIH3T3 and A549 cells, which are independent of the expression of the asialoglycoprotein receptor. Transfection activity of Gal-alpha-CDE conjugate (DSG 4) was insensitive to the existence of competitors (asialofetuin and galactose) and serum. In addition, no cytotoxicity after transfection of the complex of pDNA with Gal-alpha-CDE conjugate (DSG 4) was observed. These results suggest the potential use of Gal-alpha-CDE conjugate (DSG 4) as a non-viral vector in various cells.