PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (268)
 

Clipboard (0)
None
Journals
more »
Year of Publication
1.  The Evolving Genome of Salmonella enterica Serovar Pullorum 
Journal of Bacteriology  2002;184(10):2626-2633.
Salmonella enterica serovar Pullorum is a fowl-adapted bacterial pathogen that causes dysentery (pullorum disease). Host adaptation and special pathogenesis make S. enterica serovar Pullorum an exceptionally good system for studies of bacterial evolution and speciation, especially regarding pathogen-host interactions and the acquisition of pathogenicity. We constructed a genome map of S. enterica serovar Pullorum RKS5078, using I-CeuI, XbaI, AvrII, and SpeI and Tn10 insertions. Pulsed-field gel electrophoresis was employed to separate the large DNA fragments generated by the endonucleases. The genome is 4,930 kb, which is similar to most salmonellas . However, the genome of S. enterica serovar Pullorum RKS5078 is organized very differently from the majority of salmonellas, with three major inversions and one translocation. This extraordinary genome structure was seen in most S. enterica serovar Pullorum strains examined, with different structures in a minority of S. enterica serovar Pullorum strains. We describe the coexistence of different genome structures among the same bacteria as genomic plasticity. Through comparisons with S. enterica serovar Typhimurium, we resolved seven putative insertions and eight deletions ranging in size from 12 to 157 kb. The genomic plasticity seen among S. enterica serovar Pullorum strains supported our hypothesis about its association with bacterial evolution: a large genomic insertion (157 kb in this case) disrupted the genomic balance, and rebalancing by independent recombination events in individual lineages resulted in diverse genome structures. As far as the structural plasticity exists, the S. enterica serovar Pullorum genome will continue evolving to reach a further streamlined and balanced structure.
doi:10.1128/JB.184.10.2626-2633.2002
PMCID: PMC135040  PMID: 11976291
2.  NOVEL COMPLEX INTEGRATING MITOCHONDRIA AND THE MICROTUBULAR CYTOSKELETON WITH CHROMOSOME REMODELING AND TUMOR SUPPRESSOR RASSF1 DEDUCED BY IN SILICO HOMOLOGY ANALYSIS, INTERACTION CLONING IN YEAST, AND COLOCALIZATION IN CULTURED CELLS 
Summary
Availability of the complete sequence of the human genome and sequence homology analysis has accelerated new protein discovery and clues to protein function. Protein–protein interaction cloning suggests multisubunit complexes and pathways. Here, we combine these molecular approaches with cultured cell colocalization analysis to suggest a novel complex and a pathway that integrate the mitochondrial location and the microtubular cytoskeleton with chromosome remodeling, apoptosis, and tumor suppression based on a novel leucine-rich pentatricopeptide repeat-motif–containing protein (LRPPRC) that copurified with the fibroblast growth factor receptor complex. One round of interaction cloning and sequence homology analysis defined a primary LRPPRC complex with novel subunits cat eye syndrome chromosome region candidate 2 (CECR2), ubiquitously expressed transcript (UXT), and chromosome 19 open reading frames 5 (C19ORF5) but still of unknown function. Immuno, deoxyribonucleic acid (DNA), and green fluorescent protein (GFP) tag colocalization analyses revealed that LRPPRC appears in both cytosol and nuclei of cultured cells, colocalizes with mitochondria and β-tubulin rather than with α-actin in the cytosol of interphase cells, and exhibits phase-dependent organization around separating chromosomes in mitotic cells. GFP–tagged CECR2B was strictly nuclear and colocalized with condensed DNA in apoptotic cells. GFP–tagged UXT and GFP–tagged C19ORF5 appeared in both cytosol and nuclei and colocalized with LRPPRC and β-tubulin. Cells exhibiting nuclear C19ORF5 were apoptotic. Screening for interactive substrates with the primary LRPPRC substrates in the human liver complementary DNA library revealed that CECR2B interacted with chromatin-associated TFIID-associated protein TAFII30 and ribonucleic acid splicing factor SRP40, UXT bridged to CBP/p300–binding factor CITED2 and kinetochore-associated factor BUB3, and C19ORF5 complexed with mitochondria-associated NADH dehydrogenase I and cytochrome c oxidase I. C19ORF5 also interacted with RASSF1, providing a bridge to apoptosis and tumor suppression.
PMCID: PMC3225227  PMID: 12762840
apoptosis; chromosome separation; cytokinesis; genetic instability; microtubule-associated proteins; nucleo-cytosolic shuttling; tumor suppressor
3.  Effect of latanoprost 0.005% and brimonidine tartrate 0.2% on pulsatile ocular blood flow in normal tension glaucoma 
The British Journal of Ophthalmology  2002;86(11):1236-1239.
Aim: To determine the effect of brimonidine tartrate 0.2% and latanoprost 0.005% on pulsatile ocular blood flow (POBF) in patients with normal tension glaucoma (NTG).
Method: NTG patients with progressive optic neuropathy, new disc haemorrhage, or field defects that threatened fixation were enrolled into a randomised, investigator masked, crossover study. Group I patients received 4 weeks each of latanoprost, lubricant, and brimonidine, while group II patients received 4 weeks each of brimonidine, lubricant, and latanoprost. Diurnal POBF was measured at baseline and after each 4 week treatment.
Results: 25 patients completed the study and had reliable POBF measurement at each visit. There was no significant diurnal change in baseline POBF (p = 0.768). Latanoprost increased POBF by 213 (SD 257) μl/min (22.8%, p <0.001) while brimonidine increased it by 97 (183) μl/min (10.4%, p = 0.014). POBF increased at 8 am (p = 0.004), 12 noon (p = 0.002), and 4 pm (p <0.001) with latanoprost, while it increased only at 8 am (p = 0.016) with brimonidine. After adjusting for the factor of IOP, neither latanoprost nor brimonidine increased POBF significantly.
Conclusions: Latanoprost increases the mean POBF that is related to its IOP lowering effect. The increase in POBF noted after brimonidine is within the range of long term variation and may not be attributable to the drug effect.
PMCID: PMC1771377  PMID: 12386077
normal tension glaucoma; haemodynamics; latanoprost; brimonidine; pulsatile ocular blood flow
4.  Quantification of glyceryl trinitrate effect through analysis of the synthesised ascending aortic pressure waveform 
Heart  2002;88(2):143-148.
Objective: To establish through analysis of the radial pressure pulse waveform the dose dependent effects of glyceryl trinitrate (GTN) on properties of different blood vessels.
Design: Radial pulse waveform was measured in randomised order before, during a five hour application of a GTN patch delivering 0.104–0.625 mg/h, and for two hours after patch removal. The radial pressure waveform (Millar applanation tonometer) was convolved into an ascending aortic wave using a generalised transfer function (SphygmoCor process) enabling measurement of aortic systolic, diastolic, pulse, mean, and augmented pressure and left ventricular ejection duration in addition to standard brachial cuff pressures.
Setting: Fu Wai and Ren Ming hospitals in Beijing, China.
Patients: 46 recumbent hospitalised patients aged 56 (9) years, awaiting electrophysiological or other diagnostic studies, fasting, and with other treatments suspended.
Major outcome measures: Conventional brachial pressure measures and data from the synthesised aortic pulse.
Results: There was no consistent change in heart rate or brachial pressures except for a decrease in systolic and pulse pressures (p < 0.01) at dose > 0.416 mg/h. In contrast, there were substantial and significant (p < 0.0001) decreases in aortic systolic, pulse, and augmented pressures at all doses, mean pressure (p < 0.001) at doses > 0.416 mg/h, and ejection duration (p < 0.001) at doses > 0.208 mg/h.
Conclusions: Pulse waveform analysis exposes dose dependent effects of GTN on the aortic waveform, suggesting muscular conduit arterial dilatation with reduced wave reflection at the lowest dose, arteriolar dilatation and decreased peripheral resistance at the highest dose, and venous dilatation at the intermediate dose.
PMCID: PMC1767201  PMID: 12117838
glyercyl trinitrate; pulsed waveform analysis; aortic pressure waveform
6.  Effects ofcis-9,trans-11-conjugated linoleic acid on cancer cell cycle 
Objectives
To determine the effect of cis-9, trans-11-conjugated linoleic acid on the cell cycle of mammary cancer cells (MCF-7) and its possible mechanism of inhibition cancer growth.
Methods
Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B1, D1, p16ink4a and p21cip/wafl of MCF-7 cells which were treated with various c9, t11-CLA concentrations (25 mM, 50 mM, 100 mM and 200 mM) of c9, t11-CLA for 24 and 48 h, with negative controls (0.1% ethanol).
Results
The cell growth and DNA synthesis of MCF-7 cells were inhibited by c9, t11-CLA. MCF-7 cells, after treatment with various c9, t11-CLA doses mentioned above for 8 days, the inhibition frequency was 27.18%, 35.43%, 91.05%, and 92.86%, respectively and the inhibitory effect of c9, t11-CLA on DNA synthesis (except for 25 mM, 24 h) incorporated significantly less3H-TdR than did the negative control (P<0.05 andP<0.01). To further investigate the influence on the cell cycle progression, we found that c9, t11-CLA may arrest the cell cycle of MCF-7 cells. Immunocytochemical staining demonstrated that MCF-7 cells preincubated in media supplemented with different c9, t11-CLA concentrations at various times significantly decreased the expressions of PCNA, and Cyclin, A, B1, D1 compared with the negative controls (P<0.01), whereas the expressions of p16ink4a and p21cip/wafl, cyclin-dependent kinases inhibitors (CDKI), were increased.
Conclusions
The cell growth and proliferation of MCF-7 cells is inhibited by c9, t11-CLA by blocking the cell cycle, which reduces expressions of cyclin A, B1, D1 and enhances expressions of CDKI (p16ink4a and p21cip/wafl).
doi:10.1007/BF02898006
PMCID: PMC2723588  PMID: 21432279
mammary adenocarcinoma cells (MCF-7); cis-9; trans-11-conjugated linoleic acid (c9, t11-CLA); immunocytochemistry; cell cycle; inhibition
7.  Virus Replication and Cytokine Production in Dengue Virus-Infected Human B Lymphocytes 
Journal of Virology  2002;76(23):12242-12249.
Dengue virus (DV) replication, antibody-enhanced viral infection, and cytokine responses of human primary B lymphocytes (cells) were characterized and compared with those of monocytes. The presence of a replication template (negative-strand RNA intermediate), viral antigens including core and nonstructural proteins, and increasing amounts of virus with time postinfection indicated that DV actively replicated in B cells. Virus infection also induced B cells to produce interleukin-6 and tumor necrosis factor alpha, which have been previously implicated in virus pathogenesis. In addition, a heterologous antibody was able to enhance both virus and cytokine production in B cells. Furthermore, the levels of virus replication, antibody-enhanced virus replication, and cytokine responses observed in B cells were not statistically different from those in monocytes. These results suggest that B cells may play an important role in DV pathogenesis.
doi:10.1128/JVI.76.23.12242-12249.2002
PMCID: PMC136880  PMID: 12414963
8.  Perinatal Blockade of B7-1 and B7-2 Inhibits Clonal Deletion of Highly Pathogenic Autoreactive T Cells 
A number of in vitro studies have suggested that costimulatory molecules B7-1 and B7-2 and their receptor CD28 can promote clonal deletion, and limited in vivo studies have indicated that CD28 is involved in the clonal deletion of some T cells. However, the significance of B7-mediated clonal deletion in preventing autoimmune diseases has not been studied systematically. Here we report that the perinatal blockade of B7-1 and B7-2 substantially inhibits the clonal deletion of T cells in the thymus and leads to an accumulation of T cells capable of inducing fatal multiorgan inflammation. These results reveal a critical role for costimulatory molecules B7-1 and B7-2 in deleting pathogenic autoreactive T cells in the thymus. The critical role of B7-1 and B7-2 in T cell clonal deletion may explain, at least in part, the paradoxical increase of autoimmune disease in mice deficient for this family of costimulatory molecules, such as cytotoxic T lymphocyte associated molecule 4, CD28, and B7-2. The strong pathogenicity of the self-reactive T cells supports a central hypothesis in immunology, which is that clonal deletion plays an important role in preventing autoimmune diseases.
doi:10.1084/jem.20011948
PMCID: PMC2193695  PMID: 11956287
autoimmunity; clonal deletion; B7-1 and B7-2; CD28; CTLA-4
9.  Improved Hepatic Gene Transfer by Using an Adeno-Associated Virus Serotype 5 Vector 
Journal of Virology  2002;76(20):10497-10502.
Adeno-associated viral (AAV) vectors have been shown to direct stable gene transfer and expression in hepatocytes, which makes them attractive tools for treatment of inherited disorders such as hemophilia B. While substantial levels of coagulation factor IX (F.IX) have been achieved using AAV serotype 2 vectors, use of a serotype 5 vector further improves transduction efficiency and levels of F.IX transgene expression by 3- to 10-fold. In addition, the AAV-5 vector transduces a higher proportion of hepatocytes (∼15%). The subpopulations of hepatocytes transduced with either vector widely overlap, with the AAV-5 vector transducing additional hepatocytes and showing a wider area of transgene expression throughout the liver parenchyma.
doi:10.1128/JVI.76.20.10497-10502.2002
PMCID: PMC136579  PMID: 12239326
10.  Maximal Induction of a Subset of Interferon Target Genes Requires the Chromatin-Remodeling Activity of the BAF Complex 
Molecular and Cellular Biology  2002;22(18):6471-6479.
The mammalian SWI/SNF-like chromatin-remodeling BAF complex plays several important roles in controlling cell proliferation and differentiation. Interferons (IFNs) are key mediators of cellular antiviral and antiproliferative activities. In this report, we demonstrate that the BAF complex is required for the maximal induction of a subset of IFN target genes by alpha IFN (IFN-α). The BAF complex is constitutively associated with the IFITM3 promoter in vivo and facilitates the chromatin remodeling of the promoter upon IFN-α induction. Furthermore, we show that the ubiquitous transcription activator Sp1 interacts with the BAF complex in vivo and augments the BAF-mediated activation of the IFITM3 promoter. Sp1 binds constitutively to the IFITM3 promoter in the absence of the BAF complex, suggesting that it may recruit and/or stabilize the BAF complex binding to the IFITM3 promoter. Our results bring new mechanistic insights into the antiproliferative effects of the chromatin-remodeling BAF complex.
doi:10.1128/MCB.22.18.6471-6479.2002
PMCID: PMC135632  PMID: 12192045
11.  Label-free hybridization detection of a single nucleotide mismatch by immobilization of molecular beacons on an agarose film 
Nucleic Acids Research  2002;30(12):e61.
We developed a new technique to immobilize a set of molecular beacons on an agarose film-coated slide and found that it has the ability to identify a single nucleotide difference in label-free DNA targets. The annealing properties, specificity and hybridization dynamics of the present technique were compared with those of the conventional technique that directly immobilizes molecular beacons on a planar glass slide. It is demonstrated that the molecular beacon array on an agarose film has high quench efficiency, an excellent discrimination ratio for single nucleotide mismatches and a short detection time. We hypothesize that such a low fluorescence background and high specificity molecular beacon array will find practical applications in label-free, high-throughput mutation analysis and disease diagnosis.
PMCID: PMC117303  PMID: 12060699
12.  Low-cost virtual instrumentation system of an energy-dispersive X-ray spectrometer for a scanning electron microscope 
The paper describes an energy-dispersive X-ray spectrometer for a scanning electron microscope (SEM-EDXS). It was constructed using the new architecture of a virtual instrument (VI), which is low-cost, space-saving, fast and flexible way to develop the instrument. Computer-aided teaching (CAT) was used to develop the instrument and operation rather than a traditional instrument technique. The VI was designed using the object-oriented program language C++ and compact programmable logical devices (CPLD). These include spectra collection and processing, quantitative analysis and X-ray-intensity distribution analysis. The procedure is described in detail. The VI system gives an e¡ective and user-friendly human interface for the whole analytical task. Some examples are described.
doi:10.1155/S1463924602000160
PMCID: PMC2562962  PMID: 18924732
13.  Synthesis and Biological Activity of Organothiophosphoryl Polyoxotungstates 
Metal-Based Drugs  2002;8(5):257-262.
Organothiophosphoryl polyoxotungstates R∋XW∞∞O∋∃/- , R∋ P∋W∞,O∞/-, R∋PW∃O∋ Δ-(X = P, Si, Ge, B or Ga; R = PhP(S), C6H11P(S)) have been prepared from lacunary polyoxoanions and PhP(S). The products were characterized by elemental analysis, IR, and NMR spectroscopy. According to spectroscopic observations, the hybrid anions consist of a lacunary anion framework on which are grafted two equivalent or groups through P-O-W bridges. Some of the title compounds showed the antigerm activity.
doi:10.1155/MBD.2002.257
PMCID: PMC2365281  PMID: 18476003
14.  IGFBP-3 mediates p53-induced apoptosis during serum starvation 
International journal of oncology  2002;21(2):327-335.
Insulin-like growth factor binding protein (IGFBP)-3, a p53-response gene, can induce apoptosis in an IGF-independent manner. Here we demonstrate that IGFBP-3 mediates p53-induced apoptosis during serum starvation using two foil neoplastic cell models: one which introduces p53 activity and one which eliminates it. We created a doxycycline-inducible p53 model from the p53-negative PC-3 prostate cancer cell line. Doxycycline treatment increased both p53 and IGFBP-3 levels. It also augmented apoptosis, but not during insulin-like growth factor-I co-treatment. In a second model, lung carcinoma H460 cells expressing fully functional p53 were stably transfected with E6, which targets p53 for degradation. H460-E6 cells contained less p53 and IGFBP-3 than control neo-transfected cells, and proteasome blockade restored both. In serum deprivation, H460-E6 cells had enhanced growth and less apoptosis than did H460-neo cells. Reductions in H460-neo apoptosis, comparable in magnitude to H460-E6, were achieved by adding anti-IGFBP-3-antibody or IGFBP-3 antisense oligomers, but not non-specific immunoglobulin or IGFBP-3 sense oligomers. In summary, turning p53 ‘on’ in two foil neoplastic cell models induced IGFBP-3 expression and increased apoptosis during serum starvation, an effect inhibited by insulin-like growth factor-I treatment and specific IGFBP-3 blockade. This is the first demonstration of inhibition of p53 action by antagonizing IGFBP-3.
PMCID: PMC4152903  PMID: 12118329
IGFBP-3; p53; apoptosis; serum starvation
15.  Endoproteolytic Activity of the Proteasome 
Science (New York, N.Y.)  2002;299(5605):408-411.
The proteasome plays a central role in the degradation of regulatory and misfolded proteins. Current models suggest that substrates access the internal catalytic sites by processively threading their termini through the gated substrate channel. Here, we found that latent (closed) and activated (open) proteasomes degraded two natively disordered substrates at internal peptide bonds even when they lacked accessible termini, suggesting that these substrates themselves promoted gating of the proteasome. This endoproteolysis provides a molecular mechanism for regulated release of transcription factors from inactive precursors as well as a means of accessing internal folding defects of misfolded multidomain proteins.
doi:10.1126/science.1079293
PMCID: PMC3516294  PMID: 12481023
16.  Sequence Analysis of LRPPRC and Its SEC1 Domain Interaction Partners Suggests Roles in Cytoskeletal Organization, Vesicular Trafficking, Nucleocytosolic Shuttling and Chromosome Activity 
Genomics  2002;79(1):124-136.
LRPPRC (originally called LRP130) is an intracellular 130-kDa leucine-rich protein that co-purifies with the FGF receptor from liver cell extracts and has been detected in diverse multi-protein complexes from the cell membrane, cytoskeleton and nucleus. Here we report results of a sequence homology analysis of LRPPRC and its SEC1 domain interactive partners. Twenty-three copies of tandem repeats that are similar to PPR, TPR and HEAT repeats characterize the LRPPRC sequence. The N-terminus exhibits multiple copies of leucine-rich nuclear transport signals followed by ENTH, DUF28 and SEC1 homology domains. We used the SEC1 domain to trap interactive partners expressed from a human liver cDNA library. Interactive C19ORF5 (XP_038600) exhibited a strong homology to microtubule-associated proteins (MAP) and a potential arginine-rich mRNA binding motif. UXT (XP_033860) exhibited α-helical properties homologous to the actin-associated spectrin repeat and L/I heptad repeats in mobile transcription factors. C6ORF34 (XP_004305) was homologous to the non-DNA binding C-terminus of the E. coli Rob transcription factor. CECR2 (AAK15343) exhibited a transcription factor AT-hook motif next to two bromodomains and a homology to guanylate-binding protein 1. Taken together these features suggest a regulatory role of LRPPRC and its SEC1 domain-interactive partners in integration of cytoskeletal networks with vesicular trafficking, nucleocytosolic shuttling, chromosome remodeling and transcription.
doi:10.1006/geno.2001.6679
PMCID: PMC3241999  PMID: 11827465
FGF receptor; LRP repeat; microtubule-associated proteins; CECR2; chromosome remodeling; exocytosis; secretion; mobile transcription factors
17.  Quantitative Analysis of Acute Myocardial Infarct in Rat Hearts with Ischemia–Reperfusion Using a High-Resolution Stationary SPECT System 
The purpose of this study was to develop an in vivo imaging protocol for a high-resolution stationary SPECT system, called FASTSPECT, in a rat heart model of ischemia–reperfusion (IR) and to compare 99mTc-sestamibi imaging and triphenyltetrazolium chloride (TTC) staining for reliability and accuracy in the measurement of myocardial infarcts.
Methods
FASTSPECT consists of 24 modular cameras and a 24-pinhole aperture with 1.5-mm spatial resolution and 13.3 cps/μCi (0.359 cps/kBq) sensitivity. The IR heart model was created by ligating the left coronary artery for 90 min and then releasing the ligature for 30 min. Two hours after 99mTc-sestamibi injection (5–10 mCi [185–370 MBq]), images were acquired for 5–10 min for 5 control rats and 11 IR rats. The hearts were excised, and the left ventricle was sectioned into 4 slices for TTC staining.
Results
Left and right ventricular myocardium in control rats was shown clearly, with uniform 99mTc-sestamibi distribution and 100% TTC staining for viable myocardium. Nine of 11 rats with IR survived throughout imaging and exhibited 50.8% ± 2.7% ischemic area and 37.9% ± 3.9% infarct in the left ventricle on TTC staining. The infarct size measured by FASTSPECT imaging was 37.6% ± 3.6%, which correlated significantly with that measured by TTC staining (r = 0.974; P < 0.01).
Conclusion
The results confirmed the accuracy of FASTSPECT imaging for measurement of acute myocardial infarcts in rat hearts. Application of FASTSPECT imaging in small animals may be feasible for investigating myocardial IR injury and the effects of revascularization.
PMCID: PMC3062997  PMID: 12097466
high-resolution SPECT; 99mTc-sestamibi; myocardial infarction; ischemia–reperfusion
18.  Alternative Splicing of an Insect Sodium Channel Gene Generates Pharmacologically Distinct Sodium Channels 
Alternative splicing is a major mechanism by which potassium and calcium channels increase functional diversity in animals. Extensive alternative splicing of the para sodium channel gene and developmental regulation of alternative splicing have been reported in Drosophila species. Alternative splicing has also been observed for several mammalian voltage-gated sodium channel genes. However, the functional significance of alternative splicing of sodium channels has not been demonstrated. In this study, we identified three mutually exclusive alternative exons encoding part of segments 3 and 4 of domain III in the German cockroach sodium channel gene, paraCSMA. The splice site is conserved in the mouse, fish, and human Nav1.6 sodium channel genes, suggesting an ancient origin. One of the alternative exons possesses a stop codon, which would generate a truncated protein with only the first two domains. The splicing variant containing the stop codon is detected only in the PNS, whereas the other two full-size variants were detected in both the PNS and CNS. When expressed in Xenopus oocytes, the two splicing variants produced robust sodium currents, but with different gating properties, whereas the splicing variant with the stop codon did not produce any detectable sodium current. Furthermore, these two functional splicing variants exhibited a striking difference in sensitivity to a pyrethroid insecticide, deltamethrin. Exon swapping partially reversed the channel sensitivity to deltamethrin. Our results therefore provide the first evidence that alternative splicing of a sodium channel gene produces pharmacologically distinct channels.
PMCID: PMC3062512  PMID: 12097481
alternative splicing; para; paraCSMA; sodium channel; pyrethroid insecticide; Xenopus oocyte expression system
19.  Novel sodium channel gene mutations in Blattella germanica reduce the sensitivity of expressed channels to deltamethrin 
Pyrethroid insecticides alter the normal gating of voltage-gated sodium channels in the nervous system. Three sodium channel mutations (E434K, C764R, L993F) were recently identified in pyrethroid resistant German cockroach populations. In this report, we show that the L993F mutation decreased sodium channel sensitivity to the pyrethroid, deltamethrin, by five-fold in Xenopus oocytes. In contrast, neither E434K nor C764R alone decreased channel sensitivity to deltamethrin. However, E434K or C764R combined with L993F reduced deltamethrin sensitivity by 100-fold. Furthermore, concomitant presence of all three mutations (KRF) reduced channel sensitivity to deltamethrin by 500-fold. None of the mutations significantly affected channel gating. However, sodium current amplitudes from the mutant sodium channel carrying either E434K or C764R alone were much reduced compared to those of the wild-type channel or the channel carrying the double or triple mutations (KF, RF and KRF). These results indicated that evolution of sodium channel insensitivity in the German cockroach is achieved by sequential selection of a primary mutation L993F and two secondary mutations E434K and C764R, and concomitant presence of all three mutations dramatically reduced sodium channel sensitivity to deltamethrin.
PMCID: PMC3057061  PMID: 11886779
Knockdown resistance; Pyrethroids; Insecticide resistance; Sodium channel; Xenopus oocyte expression system
20.  Synergistic interaction between two cockroach sodium channel mutations and a tobacco budworm sodium channel mutation in reducing channel sensitivity to a pyrethroid insecticide 
Pyrethroid insecticide resistance due to reduced nerve sensitivity, known as knockdown resistance (kdr or kdr-type), is linked to multiple point mutations in the para-homologous sodium channel genes. Previously we demonstrated that two mutations (E434K and C764R) in the German cockroach sodium channel greatly enhanced the ability of the L993F mutation (a known kdr -type mutation) to reduce sodium channel sensitivity to deltamethrin, a pyrethroid insecticide. Neither E434K nor C764R alone, however, altered sodium channel sensitivity. To examine whether E434K and C764R also enhance the effect of pyrethroid resistance-associated sodium channel mutations identified in other insects, we introduced a V to M mutation (V409M) into the cockroach sodium channel protein at the position that corresponds to the V421M mutation in the Heliothis virescens sodium channel protein. We found that the V409M mutation alone modified the gating properties of the sodium channel and reduced channel sensitivity to deltamethrin by 10-fold. Combining the V409M mutation with either the E434K or C764K alone did not reduce the V409M channel sensitivity to deltamethrin further. However, the triple mutation combination (V409M, E434K and C764R) dramatically reduced channel sensitivity by 100-fold compared with the wild-type channel. These results suggest that the E434K and C764R mutations are important modifiers of sodium channel sensitivity to pyrethroid insecticides.
PMCID: PMC3049304  PMID: 11886774
Knockdown resistance; Pyrethroids; Insecticide resistance; Sodium channel; Xenopus oocyte expression system
21.  Involvement of Both Gq/11 and Gs Proteins in Gonadotropin-releasing Hormone Receptor-mediated Signaling in LβT2 Cells* 
The Journal of biological chemistry  2002;277(35):32099-32108.
The hypothalamic hormone gonadotropin-releasing hormone (GnRH) stimulates the synthesis and release of the pituitary gonadotropins. GnRH acts through a plasma membrane receptor that is a member of the G protein-coupled receptor (GPCR) family. These receptors interact with heterotrimeric G proteins to initiate downstream signaling. In this study, we have investigated which G proteins are involved in GnRH receptor-mediated signaling in LβT2 pituitary gonadotrope cells. We have shown previously that GnRH activates ERK and induces the c-fos and LHβ genes in these cells. Signaling via the Gi subfamily of G proteins was excluded, as neither ERK activation nor c-Fos and LHβ induction was impaired by treatment with pertussis toxin or a cell-permeable peptide that sequesters Gβγ-subunits. GnRH signaling was partially mimicked by adenoviral expression of a constitutively active mutant of Gαq (Q209L) and was blocked by a cell-permeable peptide that uncouples Gαq from GPCRs. Furthermore, chronic activation of Gαq signaling induced a state of GnRH resistance. A cell-permeable peptide that uncouples Gαs from receptors was also able to inhibit ERK, c-Fos, and LHβ, indicating that both Gq/11 and Gs proteins are involved in signaling. Consistent with this, GnRH caused GTP loading on Gs and Gq/11 and increased intracellular cAMP. Artificial elevation of cAMP with forskolin activated ERK and caused a partial induction of c-Fos. Finally, treatment of Gαq (Q209L)-infected cells with forskolin enhanced the induction of c-Fos showing that the two pathways are independent and additive. Taken together, these results indicate that the GnRH receptor activates both Gq and Gs signaling to regulate gene expression in LβT2 cells.
doi:10.1074/jbc.M203639200
PMCID: PMC2930616  PMID: 12050161
22.  Nucleic acid evolution and minimization by nonhomologous random recombination 
Nature biotechnology  2002;20(10):1024-1029.
We have developed a simple method for exploring nucleic acid sequence space by nonhomologous random recombination (NRR) that enables DNA fragments to randomly recombine in a length-controlled manner without the need for sequence homology. We compared the results of using NRR and error-prone PCR to evolve DNA aptamers that bind streptavidin. Starting with two parental sequences of modest avidin affinity, evolution using NRR resulted in aptamers with 15- to 20-fold higher affinity than the highest-affinity aptamers evolved using error-prone PCR, and 27- or 46-fold higher affinities than parental sequences derived using systematic evolution of ligands by exponential enrichment (SELEX). NRR also facilitates the identification of functional regions within evolved sequences. Inspection of a small number of NRR-evolved clones identified a 40-base DNA sequence, present in multiple copies in each clone, that binds streptavidin. Our findings suggest that NRR may enhance the effectiveness of nucleic acid evolution and the ease of identifying structure–activity relationships among evolved sequences.
doi:10.1038/nbt736
PMCID: PMC2819268  PMID: 12219078
23.  A 3-Dimensional Absorbed Dose Calculation Method Based on Quantitative SPECT for Radionuclide Therapy: Evaluation for 131I Using Monte Carlo Simulation 
A general method is presented for patient-specific 3-dimensional absorbed dose calculations based on quantitative SPECT activity measurements.
Methods
The computational scheme includes a method for registration of the CT image to the SPECT image and position-dependent compensation for attenuation, scatter, and collimator detector response performed as part of an iterative reconstruction method. A method for conversion of the measured activity distribution to a 3-dimensional absorbed dose distribution, based on the EGS4 (electron-gamma shower, version 4) Monte Carlo code, is also included. The accuracy of the activity quantification and the absorbed dose calculation is evaluated on the basis of realistic Monte Carlo–simulated SPECT data, using the SIMIND (simulation of imaging nuclear detectors) program and a voxel-based computer phantom. CT images are obtained from the computer phantom, and realistic patient movements are added relative to the SPECT image. The SPECT-based activity concentration and absorbed dose distributions are compared with the true ones.
Results
Correction could be made for object scatter, photon attenuation, and scatter penetration in the collimator. However, inaccuracies were imposed by the limited spatial resolution of the SPECT system, for which the collimator response correction did not fully compensate.
Conclusion
The presented method includes compensation for most parameters degrading the quantitative image information. The compensation methods are based on physical models and therefore are generally applicable to other radionuclides. The proposed evaluation methodology may be used as a basis for future intercomparison of different methods.
PMCID: PMC2804897  PMID: 12163637
Monte Carlo; absorbed dose; SPECT; 131I; image registration
24.  The Association Between Neuronal Nitric Oxide Synthase and Neuronal Sensitivity in the Brain After Brain Injury 
Injury to the central nervous system is the leading cause of disability in the United States. Neuronal death is one of the causes of disability. Among patients who survive this type of injury, various degrees of recovery in brain function are observed. The molecular basis of functional recovery is poorly understood. Clinical observations and research using experimental injury models have implicated several metabolites in the cascade of events that lead to neuronal degeneration. The levels of intracellular ATP (energy source) and pH are decreased, whereas levels of extracellular glutamate, intracellular calcium ions, and oxidative damage to RNA/DNA, protein, and lipid are increased. These initiating events can be associated with energy failure and mitochondrial dysfunction, resulting in functional or structural brain damage. The injured brain is known to express immediate early genes. Recent studies show that reactive oxygen species (ROS) cause lesions in genes from which mRNA is transcribed as part of the endogenous neuroprotective response. Although degenerating proteins and lipids may contribute to necrosis significantly after severe injury, abnormalities in genetic material, if not repaired, disturb cellular function at every level by affecting replication, transcription, and translation. These lesions include abnormal nucleic acids, known as oxidative lesions of DNA (ODLs) or of RNA (ORLs). In this review, we focus on our current understanding of the various effects of neuronal nitric oxide synthase on the formation of modified bases in DNA and RNA that are induced in the brain after injury, and how ODLs and ORLs affect cell function.
PMCID: PMC2751793  PMID: 12076978
oxidative stress; immediate early gene; gene repair; 8-oxo-guanosine glycosylase/AP lyase; Fos/AP-1; stroke; neuroprotection; nNOS; head injury; cerebral ischemia-reperfusion injury
25.  Homogeneous repair of nuclear genes after experimental stroke 
Journal of neurochemistry  2002;80(1):111-118.
The repair of oxidative DNA lesions (ODLs) in the nucleus of ischemic cortical brain cells was examined following experimentally induced stroke by occluding the right middle cerebral artery and both common carotid arteries for 60–90 min followed by reperfusion in male long-Evans hooded rats. The control group consisted of sham-operated animals undergoing the same surgery without vessel occlusion. Using a gene-specific assay based upon the presence of Escherichia coli Fpg protein-sensitive sites, we noted that animals with stroke exhibited six and four ODLs per gene in the actin and DNA polymerase-β genes, respectively. This was increased from one per four copies of each gene in the sham-operated control (p < 0.01). One half of the initial ODLs was repaired within 30 min, and 83% of them were repaired as early as 45 min of reperfusion. There was no further increase when gene repair was measured again at 2 h of reperfusion. The rates of active repair within 45 min of reperfusion were the same in these two genes (p = 0.103, anova). BrdU (10 mg/kg) was administered via intraperitoneal injection at least one day before surgery. We observed that there was no significant incorporation of BrdU triphosphates into genomic DNA during active repair, but there were significant amounts of BrdU triphosphate in nuclear DNA after active repair. The result indicates that genomic repair of ODLs in the brain did not significantly incorporate BrdU, and the initiation of neurogenesis probably starts after the completion of repair in the brain.
PMCID: PMC2729788  PMID: 11796749
brain repair; bromodeoxyuridine; cerebral ischemia-reperfusion; neurogenesis; oxidative stress; stroke

Results 1-25 (268)