Differential DNA methylation of steatosis and non-alcoholic fatty liver disease in adolescence.

BACKGROUND AND AIMS: Epigenetic modifications are associated with hepatic fat accumulation and non-alcoholic fatty liver disease (NAFLD). However, few epigenetic modifications directly implicated in such processes have been identified during adolescence, a critical developmental window where physiological changes could influence future disease trajectory. To investigate the association between DNA methylation and NAFLD in adolescence, we undertook discovery and validation of novel methylation marks, alongside replication of previously reported marks. APPROACH AND RESULTS: We performed a DNA methylation epigenome-wide association study (EWAS) on DNA from whole blood from 707 Raine Study adolescents phenotyped for steatosis score and NAFLD by ultrasound at age 17. Next, we performed pyrosequencing validation of loci within the most 100 strongly associated differentially methylated CpG sites (dmCpGs) for which ≥ 2 probes per gene remained significant across four statistical models with a nominal p value < 0.007. EWAS identified dmCpGs related to three genes (ANK1, MIR10a, PTPRN2) that met our criteria for pyrosequencing. Of the dmCpGs and surrounding loci that were pyrosequenced (ANK1 n = 6, MIR10a n = 7, PTPRN2 n = 3), three dmCpGs in ANK1 and two in MIR10a were significantly associated with NAFLD in adolescence. After adjustment for waist circumference only dmCpGs in ANK1 remained significant. These ANK1 CpGs were also associated with γ-glutamyl transferase and alanine aminotransferase concentrations. Three of twenty-two differentially methylated dmCpGs previously associated with adult NAFLD were associated with NAFLD in adolescence (all adjusted p < 2.3 × 10-3). CONCLUSIONS: We identified novel DNA methylation loci associated with NAFLD and serum liver biochemistry markers during adolescence, implicating putative dmCpG/gene regulatory pathways and providing insights for future mechanistic studies.

The millimeter-wave spectrum of the SiP radical (X2Πi): Rotational perturbations and hyperfine structure.

The millimeter/submillimeter-wave spectrum of the SiP radical (X2Πi) has been recorded using direct absorption spectroscopy in the frequency range of 151-532 GHz. SiP was synthesized in an AC discharge from the reaction of SiH4 and gas-phase phosphorus, in argon carrier gas. Both spin-orbit ladders were observed. Fifteen rotational transitions were measured originating in the Ω = 3/2 ladder, and twelve in the Ω = 1/2 substate, each exhibiting lambda doubling and, at lower frequencies, hyperfine interactions from the phosphorus nuclear spin of I = 1/2. The lambda-doublets in the Ω = 1/2 levels appeared to be perturbed at higher J, with the f component deviating from the predicted pattern, likely due to interactions with the nearby excited A2Σ+ electronic state, where ΔEΠ-Σ âˆ¼ 430 cm-1. The data were analyzed using a Hund's case aß Hamiltonian and rotational, spin-orbit, lambda-doubling, and hyperfine parameters were determined. A 2Π/2Σ deperturbation analysis was also performed, considering spin-orbit, spin-electronic, and L-uncoupling interactions. Although SiP is clearly not a hydride, the deperturbed parameters derived suggest that the pure precession hypothesis may be useful in assessing the 2Π/2Σ interaction. Interpretation of the Fermi contact term, bF, the spin-dipolar constant, c, and the nuclear spin-orbital parameter, a, indicates that the orbital of the unpaired electron is chiefly pπ in character. The bond length in the v = 0 level was found to be r0 = 2.076 Å, suggestive of a double bond between the silicon and phosphorus atoms.

Adiposity associated DNA methylation signatures in adolescents are related to leptin and perinatal factors.

Epigenetics links perinatal influences with later obesity. We identifed differentially methylated CpG (dmCpG) loci measured at 17 years associated with concurrent adiposity measures and examined whether these were associated with hsCRP, adipokines, and early life environmental factors. Genome-wide DNA methylation from 1192 Raine Study participants at 17 years, identified 29 dmCpGs (Bonferroni corrected p < 1.06E-07) associated with body mass index (BMI), 10 with waist circumference (WC) and 9 with subcutaneous fat thickness. DmCpGs within Ras Association (RalGDS/AF-6), Pleckstrin Homology Domains 1 (RAPH1), Musashi RNA-Binding Protein 2 (MSI2), and solute carrier family 25 member 10 (SLC25A10) are associated with both BMI and WC. Validation by pyrosequencing confirmed these associations and showed that MSI2 , SLC25A10 , and RAPH1 methylation was positively associated with serum leptin. These were  also associated with the early environment; MSI2 methylation (ß = 0.81, p = 0.0004) was associated with pregnancy maternal smoking, SLC25A10 (CpG2 ß = 0.12, p = 0.002) with pre- and early pregnancy BMI, and RAPH1 (ß = -1.49, p = 0.036) with gestational weight gain. Adjusting for perinatal factors, methylation of the dmCpGs within MSI2, RAPH1, and SLC25A10 independently predicted BMI, accounting for 24% of variance. MSI2 methylation was additionally associated with BMI over time (17 years old ß = 0.026, p = 0.0025; 20 years old ß = 0.027, p = 0.0029) and between generations (mother ß = 0.044, p = 7.5e-04). Overall findings suggest that DNA methylation in MSI2, RAPH1, and SLC25A10 in blood may be robust markers, mediating through early life factors.

High maternal folic acid intake around conception alters mouse blastocyst lineage allocation and expression of key developmental regulatory genes.

Folate, a cofactor for the supply of one-carbon groups, is required by epigenetic processes to regulate cell lineage determination during development. The intake of folic acid (FA), the synthetic form of folate, has increased significantly over the past decade, but the effects of high periconceptional FA intake on cell lineage determination in the early embryo remains unknown. Here, we investigated the effect of maternal high FA (HFA) intake on blastocyst development and expression of key regulatory genes. C57BL/6 adult female mice were fed either Control diet (1 mg FA) for 4 weeks before conception and during the preimplantation period (Con-Con); Control diet for 4 weeks preconception, followed by HFA (5 mg FA) diet during preimplantation (Con-HFA); or HFA diet for 4 weeks preconception and during preimplantation (HFA-HFA). At E3.5, blastocyst cell number, protein, and mRNA expression were measured. In HFA-HFA blastocysts, trophectoderm cell numbers and expression of CDX2, Oct-4, and Nanog were reduced compared with Con-Con blastocysts; Con-HFA blastocysts showed lower CDX2 and Oct-4 expression than Con-Con blastocysts. These findings suggest periconceptional HFA intake induces changes in key regulators of embryo morphogenesis with potential implications for subsequent development.

Evidence for Succession and Putative Metabolic Roles of Fungi and Bacteria in the Farming Mutualism of the Ambrosia Beetle Xyleborus affinis.

The bacterial and fungal community involved in ambrosia beetle fungiculture remains poorly studied compared to the famous fungus-farming ants and termites. Here we studied microbial community dynamics of laboratory nests, adults, and brood during the life cycle of the sugarcane shot hole borer, Xyleborus affinis We identified a total of 40 fungal and 428 bacterial operational taxonomic units (OTUs), from which only five fungi (a Raffaelea fungus and four ascomycete yeasts) and four bacterial genera (Stenotrophomonas, Enterobacter, Burkholderia, and Ochrobactrum) can be considered the core community playing the most relevant symbiotic role. Both the fungal and bacterial populations varied significantly during the beetle's life cycle. While the ascomycete yeasts were the main colonizers of the gallery early on, the Raffaelea and other filamentous fungi appeared after day 10, at the time when larval hatching happened. Regarding bacteria, Stenotrophomonas and Enterobacter dominated overall but decreased in foundresses and brood with age. Finally, inferred analyses of the putative metabolic capabilities of the bacterial microbiome revealed that they are involved in (i) degradation of fungal and plant polymers, (ii) fixation of atmospheric nitrogen, and (iii) essential amino acid, cofactor, and vitamin provisioning. Overall, our results suggest that yeasts and bacteria are more strongly involved in supporting the beetle-fungus farming symbiosis than previously thought.IMPORTANCE Ambrosia beetles farm their own food fungi within tunnel systems in wood and are among the three insect lineages performing agriculture (the others are fungus-farming ants and termites). In ambrosia beetles, primary ambrosia fungus cultivars have been regarded essential, whereas other microbes have been more or less ignored. Our KEGG analyses suggest so far unknown roles of yeasts and bacterial symbionts, by preparing the tunnel walls for the primary ambrosia fungi. This preparation includes enzymatic degradation of wood, essential amino acid production, and nitrogen fixation. The latter is especially exciting because if it turns out to be present in vivo in ambrosia beetles, all farming animals (including humans) are dependent on atmospheric nitrogen fertilization of their crops. As previous internal transcribed spacer (ITS) metabarcoding approaches failed on covering the primary ambrosia fungi, our 18S metabarcoding approach can also serve as a template for future studies on the ambrosia beetle-fungus symbiosis.

The structure of ScC2 (X̃2A1): A combined Fourier transform microwave/millimeter-wave spectroscopic and computational study.

Pure rotational spectra of Sc13C2 (X̃2A1) and Sc12C13C (X̃2A') have been measured using Fourier transform microwave/millimeter-wave methods. These molecules were synthesized in a DC discharge from the reaction of scandium vapor, produced via laser ablation, with 13CH4 or 13CH4/12CH4, diluted in argon. The NKa,Kc = 10,1 → 00,0, 20,2 → 10,1, 30,3 → 20,2, and 40,4 → 30,3 transitions in the frequency range of 14 GHz-61 GHz were observed for both species, each exhibiting hyperfine splittings due to the nuclear spins of 13C (I = 1/2) and/or Sc (I = 7/2). These data have been analyzed with an asymmetric top Hamiltonian, and rotational, spin-rotation, and hyperfine parameters have been determined for Sc13C2 and Sc12C13C. In addition, a quartic force field was calculated for ScC2 and its isotopologues using a highly accurate coupled cluster-based composite method, incorporating complete basis set extrapolation, scalar relativistic corrections, outer core and inner core electron correlation, and higher-order valence correlation effects. The agreement between experimental and computed rotational constants, including the effective constant (B + C), is ∼0.5% for all three isotopologues. This remarkable agreement suggests promise in predicting rotational spectra of new transition metal-carbon bearing molecules. In combination with previous work on Sc12C2, an accurate structure for ScC2 has been established using combined experimental (B, C) and theoretical (A) rotational constants. The radical is cyclic (or T-shaped) with r(Sc-C) = 2.048(2) Å, r(C-C) = 1.272(2) Å, and ∠(C-Sc-C) = 36.2(1)°. The experimental and theoretical results also suggest that ScC2 contains a C2 - moiety and is largely ionic.

The pure rotational spectrum of the ZnBr radical (X2Σ+): Trends in the zinc halide series.

The pure rotational spectrum of ZnBr (X2Σ+) has been recorded in the frequency range 259-310 GHz using millimeter-wave direct absorption techniques. This study is the first quantitative spectroscopic investigation of this free radical. ZnBr was synthesized in a DC discharge by the reaction of zinc vapor in argon with one of three reagents: BrCH3, Br2CH2, or Br2. Eight rotational transitions were measured for six isotopologues (64Zn79Br, 64Zn81Br, 66Zn79Br, 66Zn81Br, 68Zn79Br, and 68Zn81Br), all of which exhibited spin-rotation interactions. Furthermore, transitions originating in the v = 1 through 3 excited vibrational states were obtained for certain isotopologues. Five rotational transitions were also recorded for 67Zn79Br, in which hyperfine splittings were observed arising from the 67Zn nucleus (I = 5/2). The spectra were analyzed using a Hund's case (bßJ) Hamiltonian, and rotational, spin-rotation, and 67Zn magnetic hyperfine constants were determined. Equilibrium parameters were also derived for the 64Zn79Br, 64Zn81Br, 66Zn79Br, and 66Zn81Br isotopologues, including the vibrational constant, ωe = 286 cm-1. The equilibrium bond length was derived to be re = 2.268 48(90) Å. Analysis of the 67Zn hyperfine parameters suggest a decrease in ionic character in ZnBr from the other known zinc halides, ZnF and ZnCl.

Activity pattern and personal exposure to nitrogen dioxide in indoor and outdoor microenvironments.

People are exposed to air pollution from a range of indoor and outdoor sources. Concentrations of nitrogen dioxide (NO(2)), which is hazardous to health, can be significant in both types of environments. This paper reports on the measurement and analysis of indoor and outdoor NO(2) concentrations and their comparison with measured personal exposure in various microenvironments during winter and summer seasons. Furthermore, the relationship between NO(2) personal exposure in various microenvironments and including activities patterns were also studied. Personal, indoor microenvironments and outdoor measurements of NO(2) levels were conducted using Palmes tubes for 60 subjects. The results showed significant differences in indoor and outdoor NO(2) concentrations in winter but not for summer. In winter, indoor NO(2) concentrations were found to be strongly correlated with personal exposure levels. NO(2) concentration in houses using a gas cooker was higher in all rooms than those with an electric cooker during the winter campaign, whereas there was no significant difference noticed in summer. The average NO(2) levels in kitchens with a gas cooker were twice as high as those with an electric cooker, with no significant difference in the summer period. A time-weighted average personal exposure was calculated and compared with measured personal exposures in various indoor microenvironments (e.g. front doors, bedroom, living room and kitchen); including non-smokers, passive smokers and smoker. The estimated results were closely correlated, but showed some underestimation of the measured personal exposures to NO(2) concentrations. Interestingly, for our particular study higher NO(2) personal exposure levels were found during summer (14.0+/-1.5) than winter (9.5+/-2.4).

Enhanced in vivo tumour response from combination of carboplatin and low-dose c-myc antisense oligonucleotides.

BACKGROUND: Phosphorothioate oligonucleotides ([S]ODNs) contain a modified phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used to varying success in vivo. Carboplatin is a commonly used chemotherapeutic and is associated with chemoresistance in some human tumours. The potential for combined antisense [S]ODNs and carboplatin chemotherapy has only recently been explored in vivo. MATERIALS AND METHODS: This study examines the effect of c-myc antisense oligomers delivered in isolation as naked DNA and in combination with carboplatin upon the growth kinetics of an in vivo transplantable adenocarcinoma using rodents. RESULTS: Tumours treated with a combination of 600 microg of 15-mer c-myc phosphorothioate antisense oligodeoxyribonucleotide and an intravenous administration of carboplatin (3 mg/kg), demonstrated a significant (p<0.05) retardation in tumour growth kinetics relative to a control. Two mismatch antisense controls did not significantly inhibit tumour growth. C-myc protein studies in tumour sections failed to show significant differences in c-myc expression in any of the treated tumours. CONCLUSION: This study demonstrates that carboplatin affects the relative abundance of c-myc and that combination treatment of carboplatin and c-myc phosphorothioate antisense oligonucleotides in vivo results in synergistic tumour retardation.

Methylation of CpG sites in exon 2 of the bcl-2 gene occurs in colorectal carcinoma.

BACKGROUND: Aberrant bcl-2 expression frequently occurs in colorectal carcinoma. The current study investigated if CpG sites in bcl-2 were methylated in colorectal carcinoma and if methylation correlated with loss of expression of bcl-2 mRNA. METHODS: Methylation was assessed in 23 matched normal mucosae and colonic carcinomas by Southern blotting with methylation-sensitive enzymes. Expression of bcl-2 mRNA was assessed by Northern blotting. RESULTS: A SacII site in exon 2 of the bcl-2 gene was methylated in 5 carcinomas, plus an adjacent HpaII sites in 1 tumour. SacII site in the bcl-2 promoter were not methylated. Elevated levels of bcl-2 mRNA were detected in 3 carcinomas, 5 showed decreased expression and 4 were unchanged. CONCLUSIONS: De novo methylation of CpG sites in exon 2 of the bcl-2 gene occurs during the development of colorectal carcinoma. However, there was no relationship between expression of bc1-2 mRNA and methylation of specific CpG sites.

Tumour dosimetry in human liver following hepatic yttrium-90 microsphere therapy.

Radiation dose distributions arising from intrahepatic arterial infusion of 90Y microspheres have been investigated. Tissue samples from normal liver, the tumour periphery and tumour centre were taken from a patient following infusion of 3 GBq of 32 microm diameter resin microspheres labelled with 90Y as treatment for an 80 mm diameter metastatic liver tumour. The measured microsphere distributions in three dimensions were used to calculate radiation dose patterns. Although microspheres concentrated in the tumour periphery, heterogeneous doses were delivered to all tissues. Within the tumour periphery average doses ranged from 200 Gy to 600 Gy with minimum doses between 70 Gy and 190 Gy. The average and minimum doses for the tumour centre sample were 6.8 Gy and 3.7 Gy respectively. In the normal liver sample the average dose was 8.9 Gy with a minimum dose of 5 Gy. Less than 1% of the normal liver tissue volume received more than 30 Gy, the level above which complications have resulted for whole liver exposure using external beam radiotherapy. These calculations suggest that preferential deposition of microspheres in the well-vascularized periphery of large tumours will lead to a high proportion of the tumour volume receiving a therapeutic dose, with most of the normal liver tissue being spared substantial damage.

A microsphere-liposome (microplex) vector for targeted gene therapy of cancer. II. In vivo biodistribution study in a solid tumor model.

Cationic liposomes are commonly used for transfection of plasmids into mammalian cells, while microspheres have been traditionally used for selective delivery of anticancer agents into tumor vasculature. We have developed a novel vector, comprised of cationic liposomes electrostatically bound to ion-exchange microspheres (termed 'microplex') for targeted gene therapy of solid tumors. The delivery modes tested in a rat solid tumor model were free plasmids, plasmids bound to microspheres, to liposomes, or to the combination vector. The greatest amount of chloramphenicol acetyltransferase (CAT) reporter gene expression in tumors was achieved using the microplex vector; 3.4-fold compared with free, and 1.8-fold compared with both microspherical and liposomal deliveries (p < 0.01). Tumor-to-normal kidney tissue CAT expression ratios were as follows: free 1.9:1; microspherical 3.7:1; liposomal 1.4:1 and microplexical 2.7:1. Expression between the two types of tissues was significantly different (p < 0.01) for all delivery modes. Microspheres targeted the plasmids to the tumors, while the action of cationic liposomes on cellular membranes allowed more plasmids to breach the cell membrane. This study has proven that the novel microplex vector is capable of selective delivery of genes to tumors and has the potential to target genes in clinical trials.

Analysis of the distribution of intra-arterial microspheres in human liver following hepatic yttrium-90 microsphere therapy.

The microscopic distribution of microspheres in human liver following hepatic infusion of 32 microm diameter resin microspheres labelled with 90Y as treatment for an 80 millimetre diameter liver cancer has been investigated. Microspheres were found to deposit inhomogeneously in tissues, preferentially lodging in a region approximately 6 mm wide around the periphery of the tumour. A relative concentration of microspheres of 50 to 70 times that of normal hepatic parenchyma and 65 to 94 times that in the tumour centre was measured in this region. The deposition of spheres in the tumour periphery was not uniform, and cluster analysis showed that the spheres could be classified into clusters. The number of microspheres in a cluster was skewed towards low numbers and cluster sizes varied from 20 to 1500 microm. The observed deposition patterns indicate that the vascular tumour periphery will receive much greater radiation doses from radioactive microspheres than both normal tissue and the avascular tumour centre.

Lipoplexes and tumours. A review.

The need for genotherapy to refocus its attention on to laboratory evaluation of better methods rather than proceeding to the clinic with semi-apt tools for genetic transfer has been highlighted in clinical study reports documented to date. Quintessential for tumour genotherapy is the ability to target abnormal cells, hence reducing exposure of normal cells to genetic material whilst maximizing gene dosage to tumour cells. This becomes increasingly important as genotherapy establishes itself in the clinic alongside the older modes of treatment. This review has discussed the applicability of lipoplexes for genotherapy of solid tumours. Lipoplexes have been used extensively for gene transfer into cells, such as cancerous cells, deficient for a certain gene product. While cationic liposomes have many advantages over other forms of delivery mechanisms, several problems hinder their use in-vivo. A closer examination of the physical limitations of current lipoplex preparations, the development and testing of novel formulations, combined with more attention to the cellular processes of cell membrane breaching and nuclear entry, may enhance gene delivery. Essential for tumour genotherapy is the ability to target these lipoplexes into tumour sites whilst reducing gene dosage to other normal tissues. Development of a better lipofection agent may indeed require a collaboration of the fields of physiology, cell biology, molecular biology, biochemistry, chemistry and membrane physics.

A comparison of two computer-based face identification systems with human perceptions of faces.

The performance of two different computer systems for representing faces was compared with human ratings of similarity and distinctiveness, and human memory performance, on a specific set of face images. The systems compared were a graph-matching system (Lades M, Vorbrüggen JC, Buhmann J, Lage J, von der Malsburg C, Würtz RP, Konen W. IEEE., Trans Comput 1993;42:300-311.) and coding based on principal components analysis (PCA) of image pixels (Turk M, Pentland A. J Cognitive Neurosci 1991;3:71-86.). Replicating other work, the PCA-based system produced very much better performance at recognising faces, and higher correlations with human performance with the same images, when the images were initially standardised using a morphing procedure and separate analysis of 'shape' and 'shape-free' components then combined. Both the graph-matching and (shape + shape-free) PCA systems were equally able to recognise faces shown with changed expressions, both provided reasonable correlations with human ratings and memory data, and there were also correlations between the facial similarities recorded by each of the computer models. However, comparisons with human similarity ratings of faces with and without the hair visible, and prediction of memory performance with and without alteration in face expressions, suggested that the graph-matching system was better at capturing aspects of the appearance of the face, while the PCA-based system seemed better at capturing aspects of the appearance of specific images of faces.

University performance, gender and the rural health workforce: an analysis of nursing students.

The relationship between student gender and performance was examined in first year students enrolled in the Bachelor of Nursing course at Charles Sturt University from 1991 to 1995. A greater proportion of female students achieved passing grades in subjects studied when compared to male students, irrespective of the subject discipline area. Furthermore, a significantly greater proportion of female than male students passed nursing subjects; however, no statistically significant differences in performance were detected in the proportion of male or female students in either science or humanities subjects.

Enhanced anticancer therapy mediated by specialized liposomes.

It has been a central aim of experimental and clinical therapeutics to deliver therapeutic agents as close as possible to, or if possible within, a diseased cell. Such targeting achieves two major aims of drug delivery, the maximum dose of therapeutic agent to the diseased cell and avoidance of uptake by and, usually, accompanying side-effects to normal, healthy cells. Conventional liposomes, originally used for studies in membrane biophysics and biochemistry, have been used in therapy for the past two decades. However, when applied to deliver drugs into cells, conventional liposomes proved inefficient and so novel unconventional or specialized liposomes are constantly being prepared to enhance cell-specific delivery in-vivo. One possible way of achieving better targeting is combination of the positive attributes of more than one specialized type of liposome into one vesicle. Although a limited number of studies has examined the combined effect of such dual-specialty liposomes, more studies are warranted using appropriate models. Liposomes are composed of one, a few, or many concentric bilayer membranes which alternate with aqueous spaces. The drugs are encapsulated within the aqueous internal volume if they are hydrophilic or in the lipid bilayers if they are hydrophobic (Kim 1993). Liposomes range in size from 25 nm to more than 20 microns (Sugarman & Perez-Soler 1992). Depending on their solubility and method of formulation antimicrobial, cytotoxic and other conventional drugs, hormones, antigens, enzymes, genetic material, viruses and bacteria can be incorporated in either the aqueous or hydrophobic phase. This review discusses the types and characteristics of non-conventional liposomes used in various modes of cancer therapy, mainly chemotherapy and gene therapy. It concludes with suggestions on improving these novel liposomal to effect better targeting to cancer cells.

The efficacy of doxorubicin microspheres for hepatic micrometastases in a rat tumour model.

The use of sustained-release microspheres is of potential benefit as an adjuvant treatment for patients with occult hepatic micrometastases. This study investigates the response of a model of implantable adenocarcinoma micrometastases in the livers of DA rats following the intraportal injection of doxorubicin-incorporated ion-exchange microspheres compared to free drug bolus administration. A point-counting technique was used to determine the percentage of liver consisting of tumour 13 days after treatment. This was used as an indicator of tumour response, as was the derived tumour mass. There was a significantly higher tumour response in animals treated with the microspheres compared to animals treated with free drug delivered at the same concentration. This effect, however, was shown to decrease with a delay in the time of treatment. The tumour response of the sustained-release microspheres was achieved in the absence of any detectable local or systemic toxicity. This study demonstrates the potential of sustained-release microspheres in the treatment of patients with hepatic micrometastases.

Tumor Gene Targeting Using Microspheres: Cell Culture and in vivo Studies.

In a previous study, Dass et al. (Pharm. Sci. 2:401-405, 1996), it was shown that 1 mg of two types of ion-exchange microspheres was capable of binding and releasing plasmids in a continuous-flow system to the order of 10(11) copies in phosphate-buffered saline at 37°C. However, the functionality of the plasmids was not evaluated. In this study, one of the plasmids, pCMV-CAT, was bound to both microspheres and the functionality of the biomol-ecule was examined in cell culture and in vivo transfection studies. Rat tumor cells incubated with the hydroxyapatite (HA) plasmids were transfected 5.4-fold better than when incubated with free plasmids and 56.0-fold better than polystyrene divinylbenzene (PDB) microspheres. Cells incubated with PDB microspheres were transfected 10.4-fold less than cells incubated with free plasmids. However, HA microspheres were highly cytotoxic to the cells whereas PDB spheres had no effect on cell numbers compared with control cells. Based on expression levels of delivered plasmids in the in vivo study, delivery on microspheres to kidneys was 2.7-fold better than plasmids delivered free. Microspherical delivery of plasmids to tumors was 1.6-fold better than free delivery. However, these results were not significantly different (p >. 05). The tumor/normal tissue ratio of gene expression was 4.5:1 for free delivery and 2.6-fold when delivered on microspheres. Although the difference between deliveries in the two tissues was significant (p > 0.005) for free delivery, it was not so for micro-spherical delivery. Expression of the CAT gene was not noted in either liver or spleen of any of the animals. This present study has proved that ion-exchange microspheres have no detrimental effect on released plasmid DNA expression. In an in vivo setting, resistance to enzymatic degradation of plasmids that are bound to microspheres and subsequent release of plasmids once embolization has occurred in the tumor vascular bed effected better transfection than delivery of free plasmids.