Biomedical subjects

L Wu

Publications and source records attributed to L Wu.

At least 127 records · Page 7Linked to original sources

PubMed2001

The natural killer T-cell ligand alpha-galactosylceramide prevents autoimmune diabetes in non-obese diabetic mice.

Diabetes in non-obese diabetic (NOD) mice is mediated by pathogenic T-helper type 1 (Th1) cells that arise because of a deficiency in regulatory or suppressor T cells. V alpha 14-J alpha 15 natural killer T (NKT) cells recognize lipid antigens presented by the major histocompatibility complex class I-like protein CD1d (refs. 3,4). We have previously shown that in vivo activation of V alpha 14 NKT cells by alpha-galactosylceramide (alpha-GalCer) and CD1d potentiates Th2-mediated adaptive immune responses. Here we show that alpha-GalCer prevents development of diabetes in wild-type but not CD1d-deficient NOD mice. Disease prevention correlated with the ability of alpha-GalCer to suppress interferon-gamma but not interleukin-4 production by NKT cells, to increase serum immunoglobulin E levels, and to promote the generation of islet autoantigen-specific Th2 cells. Because alpha-GalCer recognition by NKT cells is conserved among mice and humans, these findings indicate that alpha-GalCer might be useful for therapeutic intervention in human diseases characterized by Th1-mediated pathology such as Type 1 diabetes.

Animals↗

PubMed2001

Circulating levels of retinol, tocopherol and carotenoid in Nepali pregnant and postpartum women following long-term beta-carotene and vitamin A supplementation.

OBJECTIVE: To characterize circulating carotenoid and tocopherol levels in Nepali women during pregnancy and post-partum and to determine the effects of beta-carotene and vitamin A supplementation on their concentration in serum. DESIGN: Randomized community supplementation trial. SETTING: The study was carried out from 1994 to 1997 in the Southern, rural plains District of Sarlahi, Nepal. SUBJECTS: A total of 1431 married women had an ascertained pregnancy, of whom 1186 (83%) provided an analyzable serum sample during pregnancy; 1098 (77%) provided an analyzable 3-4 months post-partum serum sample. INTERVENTIONS: Women received a weekly dose of vitamin A (7000 microg RE), beta-carotene (42 mg) or placebo before, during and after pregnancy. Serum was analyzed for retinol, alpha-tocopherol, gamma-tocopherol, beta-carotene, alpha-carotene, lycopene, lutein + zeaxanthin, and beta-cryptoxanthin concentrations during mid-pregnancy and at approximately 3 months post-partum. RESULTS: Compared to placebo, serum retinol, beta-carotene, gamma-tocopherol, beta-cryptoxanthin and lutein + zeaxanthin concentrations were higher among beta-carotene recipients during pregnancy and, except for beta-cryptoxanthin, at postpartum. In the vitamin A group, serum retinol and beta-cryptoxanthin were higher during pregnancy, and retinol and gamma-tocopherol higher at postpartum. Lutein + zeaxanthin was the dominant carotenoid, regardless of treatment group, followed by serum beta-carotene. Serum lycopene level was lowest, and very low compared to the US population. Serum retinol was higher, and carotenoid and alpha-tocopherol lower, at postpartum than during pregnancy in all groups. CONCLUSIONS: Pregnant and lactating Nepali women have lower serum carotenoid and tocopherol levels than well-nourished populations. beta-carotene supplementation appeared to increase levels of tocopherol and other carotenoids in this population.

Adult↗

PubMed2001

Direct correlation between positron emission tomographic images of two reporter genes delivered by two distinct adenoviral vectors.

Biodistribution, magnitude and duration of a therapeutic transgene's expression may be assessed by linking it to the expression of a positron emission tomography (PET) reporter gene (PRG) and then imaging the PRG's expression by a PET reporter probe (PRP) in living animals. We validate the simple approach of co-administering two distinct but otherwise identical adenoviruses, one expressing a therapeutic transgene and the other expressing the PRG, to track the therapeutic gene's expression. Two PET reporter genes, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) and dopamine-2 receptor (D(2)R), each regulated by the same cytomegalovirus (CMV) promoter, have been inserted into separate adenoviral vectors (Ad). We demonstrate that cells co-infected with equivalent titers of Ad-CMV-HSV1-sr39tk and Ad-CMV-D(2)R express both reporter genes with good correlation (r(2) = 0.93). Similarly, a high correlation (r(2) = 0.97) was observed between the expression of both PRGs in the livers of mice co-infected via tail-vein injection with equivalent titers of these two adenoviruses. Finally, microPET imaging of HSV1-sr39tk and D(2)R expression with 9-(4-[(18)F]fluoro-3-hydroxymethylbutyl) guanine ([(18)F]FHBG) and 3-(2-[(18)F]fluoroethyl)spiperone ([(18)F]FESP), utilizing several adenovirus-mediated delivery routes, illustrates the feasibility of evaluating relative levels of transgene expression in living animals, using this approach.

Adenoviridae↗

PubMed2001

Chimeric PSA enhancers exhibit augmented activity in prostate cancer gene therapy vectors.

The native PSA enhancer and promoter confer prostate-specific expression when inserted into adenovirus vectors capable of efficient in vivo gene delivery, although the transcriptional activity is low. By exploiting properties of the natural PSA control regions, we have improved the activity and specificity of the prostate-specific PSA enhancer for gene therapy and imaging applications. Previous studies have established that androgen receptor (AR) molecules bind cooperatively to AREs in the PSA enhancer core (-4326 to -3935) and act synergistically with AR bound to the proximal promoter to regulate transcriptional output. To exploit the synergistic nature of AR action we generated chimeric enhancer constructs by (1) insertion of four tandem copies of the proximal AREI element; (2) duplication of enhancer core; or (3) removal of intervening sequences (-3744 to -2855) between the enhancer and promoter. By comparing to the baseline construct, PSE, containing the PSA enhancer (-5322 to -2855) fused to the proximal promoter (-541 to +12), the three most efficacious chimeric constructs, PSE-BA (insertion of ARE4), PSE-BC (duplication of core) and PSE-BAC (insertion of core and ARE4), are 7.3-, 18.9-, and 9.4-fold higher, respectively. These chimeric PSA enhancer constructs are highly androgen inducible and retain a high degree of tissue discriminatory capability. Initial biochemical studies reveal that the augmented activity of the chimeric constructs in vivo correlates with their ability to recruit AR and critical co-activators in vitro. The enhanced activity, inducibility and specificity of the chimeric constructs are retained in an adenoviral vector (Ad-PSE-BC-luc). Systemic administration of Ad-PSE-BC-luc into SCID mice harboring the LAPC-9 human prostate cancer xenografts shows that this prostate specific vector retained tissue discriminatory capability compared with a comparable cytomegalovirus (CMV) promoter driven vector.

Adenoviridae↗

PubMed2001

Adherence of multiple myeloma cells to bone marrow stromal cells upregulates vascular endothelial growth factor secretion: therapeutic applications.

Increased angiogenesis has recently been recognized in active multiple myeloma (MM). Since vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two key mediators of angiogenesis, we characterized the production of VEGF, b-FGF and interleukin-6 (IL-6) (a MM growth and survival factor) in MM cell lines and Epstein-Barr virus (EBV) transformed B cell lines from MM patients, patient MM cells, as well as bone marrow stromal cells (BMSCs) from normal healthy donors and MM patients. We detected secretion of VEGF, but no bFGF and IL-6, in MM cell lines (MM.1S, RPMI 8226 and U266); EBV transformed B cell lines from MM patients (IM-9, HS-Sultan and ARH77); MM cell lines resistant to doxorubicin (RPMI-DOX40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5) and dexamethasone (MM.1R); and patient MM cells (MM1 and MM2). BMSCs from MM patients and normal donors secreted VEGF, b-FGF and IL-6. Importantly, when MM cells were adhered to BMSCs, there was a significant increase in VEGF (1.5- to 3.1-fold) and IL-6 (1.9- to 56-fold) secretion. In contrast, the bFGF decreased in co-cultures of BMSCs and MM cells. Paraformaldehyde fixation of BMSCs or MM cells prior to adhesion revealed that VEGF was produced both from BMSCs and MM cells, though it may come primarily from BMSCs in some cultures. IL-6 was produced exclusively in BMSCs, rather than MM cells. Moreover, when MM cells were placed in Transwell insert chambers to allow their juxtaposition to BMSCs without cell to cell contact, induction of VEGF and IL-6 secretion persisted, suggesting the importance of humoral factors. Addition of exogenous IL-6 (10 ng/ml) increased VEGF secretion by BMSCs. Conversely, VEGF (100 ng/ml) significantly increased IL-6 secretion by BMSCs. Moreover, anti-human VEGF (1 microg/ml) and anti-human IL-6 (10 microg/ml) neutralizing antibodies reduced IL-6 and VEGF secretion, respectively, in cultures of BMSCs alone and co-cultures of BMSCs and MM cells. Finally, thalidomide (100 microM) and its immunomodulatory analog IMiD1-CC4047 (1 microM) decreased the upregulation of IL-6 and VEGF secretion in cultures of BMSCs, MM cells and co-cultures of BMSCs with MM cells. These data demonstrate the importance of stromal-MM cell interactions in regulating VEGF and IL-6 secretion, and suggest additional mechanisms whereby thalidomide and IMiD1-CC4047 act against MM cells in the BM millieu.

Angiogenesis Inhibitors↗

PubMed2001

Role of the Bloom's syndrome helicase in maintenance of genome stability.

The RecQ family of DNA helicases has members in all organisms analysed. In humans, defects in three family members are associated with disease conditions: BLM is defective in Bloom's syndrome, WRN in Werner's syndrome and RTS in Rothmund-Thomson syndrome. In each case, cells from affected individuals show inherent genomic instability. The focus of our work is the Bloom's syndrome gene and its product, BLM. Here, we review the latest information concerning the roles of BLM in the maintenance of genome integrity.

Adenosine Triphosphatases↗

PubMed2001

Permeant ion-dependent changes in gating of Kir2.1 inward rectifier potassium channels.

We studied the effect of monovalent thallium ion (Tl(+)) on the gating of single Kir2.1 channels, which open and close spontaneously at a constant membrane potential. In cell-attached recordings of single-channel inward current, changing the external permeant ion from K(+) to Tl(+) decreases the mean open-time by approximately 20-fold. Furthermore, the channel resides predominantly at a subconductance level, which results from a slow decay (tau = 2.7 ms at -100 mV) from the fully open level immediately following channel opening. Mutation of a pore-lining cysteine (C169) to valine abolishes the slow decay and subconductance level, and single-channel recordings from channels formed by tandem tetramers containing one to three C169V mutant subunits indicate that Tl(+) must interact with at least three C169 residues to induce these effects. However, the C169V mutation does not alter the single-channel closing kinetics of Tl(+) current. These results suggest that Tl(+) ions change the conformation of the ion conduction pathway during permeation and alter gating by two distinct mechanisms. First, they interact with the thiolate groups of C169 lining the cavity to induce conformational changes of the ion passageway, and thereby produce a slow decay of single-channel current and a dominant subconductance state. Second, they interact more strongly than K(+) with the main chain carbonyl oxygens lining the selectivity filter to destabilize the open state of the channel and, thus, alter the open/close kinetics of gating. In addition to altering gating, Tl(+) greatly diminishes Ba(2+) block. The unblocking rate of Ba(2+) is increased by >22-fold when the external permeant ion is switched from K(+) to Tl(+) regardless of the direction of Ba(2+) exit. This effect cannot be explained solely by ion-ion interactions, but is consistent with the notion that Tl(+) induces conformational changes in the selectivity filter.

Animals↗

PubMed2001

Comparing the immunogenicity of hepatitis B virus S gene variants by DNA immunization.

DNA immunization was used to compare the immunogenicity of hepatitis B virus S gene variants. Four recombinant plasmid DNAs containing the full-length virus genome with different S gene inserts were used to immunize BALB/c and C57/BL/6 mice. These inserts were cloned from 129L (residue 129, glutamine to leucine), 129H (residue 129, glutamine to histidine) 145R (residue 145, glycine to arginine) variants and the wild-type virus. The titer of hepatitis B virus core antibodies (anti-HBc) in immunized mice was used as the control for the efficiency of DNA immunization. Serum hepatitis B surface antibody (anti-HBs) titer and cytokines induced in splenocytes stimulated with hepatitis B surface antigen (HBsAg) were monitored as specific immune responses induced by different plasmid DNAs. 129L DNA induced significantly lower anti-HBs antibodies (IgG, IgG1, and IgG2a) and less interferon-gamma, compared to those in mice immunized with the 129H variant and the wild-type HBV DNA (p < 0.05). Computer modeling showed that a change from glutamine to leucine at 129 residue led to higher hydrophobicity and could result in decreased immunogenicity. Results indicate that DNA immunization can be used to compare the humoral and cellular immunogenicity among different HBV S variants.

Animals↗

PubMed2001

A novel shadow-imaging technique to measure charge distribution and lattice displacement.

We developed a novel shadow-imaging diffraction technique, using both non-coherent and coherent sources, based on parallel recording of diffraction intensity of many reflections to measure charge distribution and lattice displacement in crystals and in defects. Applying the method to Bi2Sr2CaCu2O8 superconductors demonstrated its unprecedented sensitivity and accuracy.

Journal Article↗

PubMed2001

Abnormal Ca2+ signalling in vascular endothelial cells from spontaneously hypertensive rats: role of free radicals.

OBJECTIVE: To test the hypothesis that the Ca2+ signal transduction process in endothelial cells from genetically hypertensive rats (SHR) is affected by an overproduction of free radicals. METHODS: The Ca2+ response to the inositol 1,4,5-triphosphate (IP3) mobilizing agonist, ATP, was measured using the fluorescent probe, fura-2, in endothelial cells from Sprague-Dawley rats, and in young and age-matched genetically hypertensive rats (SHR). The effect of free radicals and reducing agents on the intracellular release of Ca2+ and IP3productionwas determined in resting and ATP-stimulated cells. Experiments were also performed to compare the level of expression and enzymatic activity of catalase and superoxide dismutase (SOD) in endothelial cells from SHR and Sprague-Dawley rats. RESULTS: The exposure of aortic endothelial cells from Sprague-Dawley rats to the free-radical generating system, hypoxanthine + xanthine oxidase (HX/XO), caused a time- and concentration-dependent inhibition of the ATP-induced Ca2+ response. A similar HX/XO-dependent inhibition was also observed in Sprague-Dawley cells stimulated with the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin. Incubation with the antioxidative enzymes, catalase and SOD, had no effect on the ATP-induced Ca2+ release in Sprague-Dawley cells, but led to a strong increase in the internal release of Ca2+ in cells from adult (12 weeks old) or young (3 weeks old) SHR. The effect of antioxidants was not related either to an enhancement of the ATP-induced production of IP3, or to a lower expression and activity of SOD and catalase. CONCLUSION: The present work provides evidence that the Ca2+ signalling process in SHR endothelial cells is affected by an overproduction of free radicals, resulting in a depletion of releasable Ca2+ from IP3-sensitive and insensitive Ca2+ pools. These results point towards a beneficial action of antioxidants on Ca2+ signalling in endothelial cells from models of hypertension.

Adenosine Triphosphate↗

PubMed2001

Enhanced superoxide anion formation in vascular tissues from spontaneously hypertensive and desoxycorticosterone acetate-salt hypertensive rats.

OBJECTIVES: To investigate the basal and NADH-stimulated superoxide (.O2-) production and inactivation by Cu/Zn superoxide dismutase (SOD) in aorta from spontaneously hypertensive rats (SHR) and from desoxycorticosterone acetate (DOCA)-salt hypertensive (DOCA-HT) rats. METHODS: Tissue .O2- levels were estimated with the lucigenin-enhanced chemiluminescence method in aorta and cultured smooth muscle cells (SMCs) from SHR and in aorta from DOCA-HT rats treated for 4 weeks. RESULTS: The basal aortic .O2- generation was increased by 135 and 100%, and the NADH stimulated .O2- production was also increased 37 and 22% in SHR and in DOCA-HT rats compared to their normotensive controls, respectively. Although no difference existed in blood pressure as well as in basal and in NADH stimulated .O2- production between Wistar-Kyoto (WKY) rats and SHR rats at age of 6 weeks, O2- production and blood pressure increased concomitantly in SHR aged 9 and 12 weeks. Basal and NADH-stimulated .O2- production, in cultured SMCs, was also 80 and 64% higher, respectively, in SHR compared to WKY rats. The NADH oxidase activity was found to be increased in aorta from both SHR and DOCA-HT rats but SOD activity was reduced only in aorta from DOCA-HT rats. CONCLUSIONS: An enhanced .O2- formation resulting from an increased NADH oxidase activity was found in aorta from SHR and DOCA-HT rats. Cultured arterial SMCs from SHR also generated excessive .O2- formation under basal and stimulated conditions. The age-related increase in vascular .O2- formation in association with the rise in blood pressure in SHR suggests that the oxidative stress might contribute to the development of hypertension. NADH oxidase activity was greater in aorta of both hypertension models, but a decrease of Cu/Zn SOD activity could also contribute to the high level of aortic .O2- in DOCA-HT rats.

Aging↗

PubMed2001

Angiotensin converting enzyme inhibitor restrains inflammation-induced vascular injury in mice.

OBJECTIVES: Recent advances in molecular genetics made the mouse model important for studying the genetic basis of hypertension and vascular diseases such as the components of the renin-angiotensin system. This study was undertaken to investigate the role of angiotensin converting enzyme (ACE) in the mouse vascular injury model. DESIGN AND METHODS: Inflammation-induced vascular injury was created by placing a polyethylene cuff around the femoral artery of 12-14-week-old male FVB/N mice. Cuffed arteries were harvested and applied to reverse transcriptase-polymerase chain reaction analysis and immunohistochemistry for ACE. Subsequently, the effects of an ACE inhibitor, perindopril (3 mg/kg per day), on neointimal thickening were examined 2 weeks after cuff placement The influence of a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg per day) on the effects of perindopril was also examined. RESULTS: ACE mRNA expression increased in a time-dependent manner up to 2 weeks after cuff placement. Immunoreactive ACE was localized in the endothelium in the intact artery, while positive staining was observed in the medial and neointimal layer as well as in the periadventitial region of the cuffed artery. The intimamedia area ratio was significantly decreased by perindopril treatment (vehicle, 0.75+/-0.10; perindopril, 0.32+/-0.04; P< 0.05). The effect of perindopril was abrogated by coadministration of L-NAME whereas L-NAME alone did not affect the intima-media ratio (L-NAME, 0.66+/-0.11; perindopril + L-NAME, 0.72+/-0.09). CONCLUSIONS: This study provides evidence that ACE plays a role in cuff-induced neointimal thickening in mice. Nitric oxide may contribute, at least in part, to the inhibitory effects of perindopril.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2001

The impaired glutathione system and its up-regulation by sulforaphane in vascular smooth muscle cells from spontaneously hypertensive rats.

The glutathione (GSH) system plays an important role in reducing oxidative stress, the increase of which has been linked to the pathogenesis of hypertension. The aims of this study were to investigate: (1) whether the GSH system was impaired in aortic smooth muscle cells (SMCs) from spontaneously hypertensive rats (SHR), and (2) whether this system could be up-regulated by the phase-2 enzyme inducers, sulforaphane and t-butylhydroquinone (t-BHQ). Basal levels of cellular GSH, GSH-reductase and GSH-peroxidase were significantly lower in SMCs from SHR than from normotensive Wistar-Kyoto (WKY) rats. Heme oxygenase-1 (HO-1) was significantly higher in SHR SMCs, which correlated with the higher oxidative stress experienced by these cells. No differences were observed in the basal activity of GSH-S-transferase nor in the ability to synthesize GSH between SMCs from these two strains. Sulforaphane (0.05-1 micromol/l) and t-BHQ (10-100 micromol/l) induced significant and concentration-dependent increases in cellular GSH levels, HO-1 protein content and activities of GSH-reductase and GSH-peroxidase in SMCs from both rat strains. Upregulation of phase 2 enzymes correlated with a decrease in oxidative stress experienced by the SMCs, particularly with SHR. We conclude that SHR SMCs experience greater oxidative stress than WKY SMCs and that malfunction of the GSH system contributes to the enhanced oxidative stress in SHR SMCs.

Animals↗

PubMed2001

Microsurgical tissue transplantation or replantation in patients with psychoneurological impairment.

Sometimes patients with a psychoneurological impairment present with a traumatic injury that requires either microsurgical replantation or free-tissue transfer. We reviewed 38 patients undergoing 40 microvascular operations; the patients included 26 patients with psychological impairment (group 1), 3 with mental disability (group 2), and 9 with an acquired head injury and consciousness disturbance (Glasgow Coma Scale score < or =14) (group 3). Patients with a psychological impairment, especially those with a self-inflicted injury, are often uncooperative and do not recognize the necessity of restorative procedures. A multidisciplinary approach by the trauma surgeon, plastic surgeon, psychiatrist, and neurosurgeon, with coordinated assistance from the physician, nurse, therapist, and family, is required for treatment. In our study the success rate of replantation was 77.8 percent (14 of 18); for free tissue transfer the success rate was 95.5 percent (21 of 22). The overall success rate of microsurgical procedures (87.5 percent, 35 of 40) was similar to that in the population at large. Patients with psychological impairment tend to be lost during follow-up; therefore, their functional results may be poorer than expected. Nonetheless, patients with psychological impairment should not be deprived of the benefits of restorative surgery.

Adult↗

PubMed2001

Determination of the complex Young and shear dynamic moduli of viscoelastic materials.

The Young and shear dynamic moduli of viscoelastic materials are determined from laser vibrometric measurements of the surface motion of a three-dimensional sample excited by a piezoelectric actuator inside a chamber with controllable temperature and static pressure. The moduli are estimated from an inversion code that minimizes the difference between the data and the predictions from a finite element model in which the elastic moduli are the adjustable parameters. The technique is first used to measure the dynamic properties of homogeneous samples and the results are compared with those obtained by the standard rod resonance technique. Results are then obtained with microvoided samples in the 0.5-3 kHz frequency range, at temperatures ranging from 7 to 40 degrees C, and static pressures ranging from ambient to 34 atm (3.45 MPa or 500 psi). The limitations of the technique are discussed.

Journal Article↗

PubMed2001

Simultaneous recovery of RNA and DNA from soils and sediments.

Recovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316-322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0 degrees C to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 microg g of soil(-1) dry weight), whereas the DNA yield ranged from 23 to 435 microg g(-1). In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification.

DNA, Bacterial↗

PubMed2001

Development and evaluation of functional gene arrays for detection of selected genes in the environment.

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65 degrees C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r(2) = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.

Bacteria↗

PubMed2001

Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning.

To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three Taq DNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq (8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.

Artifacts↗