Increased risk of attention-deficit/hyperactivity disorder associated with exposure to organophosphate pesticide in Taiwanese children.

Attention-deficit/hyperactivity disorder (ADHD) is male predominated, and the etiology of this disorder remains unclear. Past studies have assessed the association of low-level organophosphate pesticide exposure with childhood ADHD cross-sectionally and prospectively. However, the results have been inconsistent. A first case-control study was performed to investigate the relationship between organophosphate pesticide exposure and ADHD with adjusted covariates. We recruited 97 doctor-diagnosed ADHD cases and 110 non-ADHD controls who were 4-15 years of age. Exposure was assessed using urinary levels of dialkylphosphate metabolites, which are biomarkers of OP pesticide exposure. Blood lead levels and polymorphisms of two commonly verified dopaminergic-related genes (the D4 dopamine receptor gene DRD4 and the dopamine transporter gene DAT1) were also analyzed. The sociodemographics and lifestyles of the children and of the mothers during pregnancy were collected using a questionnaire. The blood lead levels of both groups were similar (1.57 ± 0.73 vs. 1.73 ± 0.77 µg/dL, p = 0.15). Significant urinary concentration differences in one of the six dialkylphosphate metabolites, dimethylphosphate (DMP), were found between ADHD and control subjects (322.92 ± 315.68 vs. 224.37 ± 156.58 nmol/g cr., p < 0.01). A dose-response relationship was found between urinary concentrations of DMP and ADHD in both crude and adjusted analyses (p for trend<0.05). Children with higher urinary DMP concentrations may have a twofold to threefold increased risk of being diagnosed with ADHD. We report a dose-response relationship between child DMP levels and ADHD. Organophosphate pesticide exposure may have deleterious effects on children's neurodevelopment, particularly the development of ADHD.

CD5-mediated specific delivery of DNA to T lymphocytes: compartmentalization augmented by adenovirus.

Specific DNA delivery has been achieved via interactions between an asialoorosomucoid-polylysine conjugate and the asialoglycoprotein receptor. We have now extended this technology to another cell type. In order to achieve DNA delivery uniquely to T cells, we have employed an antibody-polylysine conjugate which binds and is internalized via CD5. Binding analyses of the T101 monoclonal antibody to Jurkat cells and freshly isolated human peripheral T lymphocytes were performed and Scatchard plots revealed Kd values of 1.4 and 1.2 pM, respectively. To introduce DNA into the T cell, a complex of T101-polylysine and the luciferase plasmid was formed (T101-PL-DNA). 125I-labeled antibody alone or T101-PL-DNA complexes were both shown to internalize. Subcellular fractionation indicated that the complex remained in the endosomal compartment of the cell for up to 90 min. However, with the addition of adenovirus particles, there was a decrease of labeled complex in the endosomal fraction over time suggesting it was no longer 'tethered' to the endosome vesicle. In vitro transfections confirmed this result showing the addition of adenovirus particles during incubation resulted in increased expression of the luciferase protein. Without adenovirus, there was limited expression of the transduced gene. These data revealed that T101 can deliver DNA via an antibody-PL conjugate. The addition of adenovirus allowed the DNA to escape the endosome enabling expression of the reporter gene.

Obstacles and advances in non-viral gene delivery.

This review focuses on recent progress and novel strategies to improve the efficiency of in vivo non-viral gene delivery. Examples of the most promising attempts to overcome specific barriers are presented in fuller detail. Current research into several of the most difficult steps in the gene delivery pathway is discussed including particle stabilization, targeting, cytoplasmic entry and access to the nucleus. The impact of recent reports on our current understanding of the true limitations to in vivo delivery is also discussed. The importance of preclinical animal models for the development of clinical applications of gene therapy is noted.

Gene therapy strategies for the treatment of chronic viral hepatitis.

Chronic viral hepatitis is a major clinical problem, with over half a billion persons infected worldwide. Current therapies, principally treatment with recombinant IFN-alpha protein, have limited benefit. Recent studies suggest that gene-based expression of IFN-alpha is a possible therapeutic alternative that may improve the effectiveness of treatment. Gene delivery to the liver and consequent IFN-alpha expression therein, has the potential to concentrate the protein at the target organ and provide more continuous exposure to the therapeutic agent. Other potential gene and nucleic acid therapeutics for viral hepatitis are also being investigated. Key to the deployment of these future therapies is a suitable method of gene delivery. Although recombinant viral vector systems, such as adenovirus, are currently the most effective means of gene delivery to the liver, their use presents many concerns. These include immune and inflammatory reactions to the viral vector and possible adverse interactions between the recombinant virus and the pre-existing viral infection. Non-viral gene delivery systems would be a preferred treatment modality. The efficiency of current non-viral systems is not adequate for systemically administered liver gene therapy. However, recent use of membrane permeabilisation techniques has shown that high efficiency non-viral gene transfer agents are possible. The future coupling of these improved delivery systems with gene- or nucleic acid-based therapeutics currently in development holds out great promise for new generations of antihepatitis therapies.

Time-resolved spectroscopy of energy transfer and trapping upon selective excitation in membranes of Heliobacillus mobilis at low temperature.

Transient absorption difference spectra in the Qy absorption band of bacteriochlorophyll (BChl) g and in the 670 nm absorption band of the primary acceptor A0 in membranes of Heliobacillus mobilis (Hc. mobilis) were measured at 20 K upon selective excitation at 668, 793, 810, and 815 nm with a 5 nm spectral bandwidth. When excited at 793 nm, the spectral equilibration of excitations from shorter to longer wavelength-absorbing pigments occurred within 3 ps and mostly localized at the band centered around 808 nm. When excited at 668 nm, the excitation energy transfer from the 670 nm absorbing pigment to the Qy band of BChl g took less than 0.5 ps, and the energy redistribution occurred and localized at 808 nm as in the case of the 793 nm excitation. All of the excitations were localized at the long wavelength pigment pool centered around 810 or 813 nm when excited at 810 or 815 nm. A slower energy transfer process with a time constant of 15 ps was also observed within the pool of long wavelength-absorbing pigments upon selective excitation at different wavelengths as has been observed by Lin et al. (Biophys. J. 1994, 67, 2479) when excited at 590 nm. Energy transfer from long wavelength antenna molecules to the primary electron donor P798 followed by the formation of P+ took place with a time constant of 55-70 ps for all excitations. Direct excitation of the primary electron acceptor A0, which absorbed at 670 nm, showed the same kinetic behavior as in the case when different forms of antenna pigments were excited in the Qy region. This observation generally supports the trapping-limited case of energy transfer in which the excitations have high escape probability from the reaction center (RC) until the charge separation takes place. Possible mechanisms to account for the apparent "uphill" energy transfer from the long wavelength antenna pigments to P798 are discussed.

Temperature dependence of charge recombination in Heliobacillus mobilis.

Transient absorption difference spectroscopy was used to study the temperature dependence of the P798+ decay kinetics in heliobacteria. For membrane samples, two components were obtained from the fitting of kinetic traces in the temperature range of 4-29 degrees C. A 3-9 ms component representing the cytochrome (cyt) c oxidation has an activation energy of 33.0 +/- 2.8 kJ/mol. A 12-22 ms component representing either P798+FX- or P798+FA/B- recombination has an activation energy of 15.3 +/- 2.4 kJ/mol. In isolated reaction centers (RC), only one 14 ms component due to P798+FX- recombination was obtained in this temperature range. The Arrhenius plot shows that the recombination rate of this P798+FX- state is temperature independent in the near room temperature range. For RC in the temperature range of 60-298 K, a 12-15 ms decay was obtained at temperatures greater than 240 K. Biphasic decay traces (12-15 ms and 2-4 ms components) were obtained at temperatures between 170 K and 230 K. Only one 2-4 ms component was found at temperatures lower than 160 K. The gradual switchover from the 12-15 ms to the 2-4 ms component upon cooling may indicate the shift of the P798+FX- recombination state to a state that is prior to P798+FX-, although other interpretations can not be excluded. The absorption difference spectrum (delta A @ 160 K - delta A @ 240 K) in the blue region shows a positive amplitude below 405 nm and a negative amplitude above 405 nm implying that the 2-4 ms decay component may be due to the recombination of P798+A1-, where A1 is a quinone-type acceptor.

Targeted delivery of DNA encoding herpes simplex virus type-1 glycoprotein D enhances the cellular response to primary viral challenge.

Intravenous injection of plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) complexed with asialoorosomucoid-poly-L-lysine (gD-ASOR) targets foreign DNA to the liver, leading to hepatic expression of gD-1. BALB/c mice were given two intravenous injections of gD-ASOR, pBK-ASOR (plasmid lacking the gD-1 gene but complexed with ASOR), or PBS. The skin was inoculated with 1 x 10(4) PFU of HSV-1 or sham-inoculated, and analyzed for infectious virus and cellular infiltration 1, 3, and 5 days after inoculation. Prior immunization with gD-ASOR led to significantly lower (P < 0.05) viral titers in the skin 5 days after inoculation compared with controls. Infiltration of the skin at the site of inoculation by polymorphonuclear neutrophils (PMNs), T cells, B cells, dendritic cells, and macrophages was monitored immunohistochemically. Significantly higher numbers (P < 0.05) of CD4+ and CD8+ T cells, dendritic cells, and macrophages responded to HSV-1 challenge in mice immunized with gD-ASOR than in mice immunized with pBK-ASOR or PBS. The response by PMNs and B cells was indistinguishable among the treatment groups. These results suggest that BALB/c mice sensitized to gD-1 following gD-ASOR immunization develop an enhanced T-cell response to primary HSV-1 infection.

Comparison of the efficacy and tolerability of telmisartan 40 mg vs. enalapril 10 mg in the treatment of mild-to-moderate hypertension: a multicentre, double-blind study in Taiwanese patients.

The purpose of this randomised, double-blind, double-dummy, parallel-group study was to evaluate the efficacy and tolerability of telmisartan 40 mg once daily vs. enalapril 10 mg once daily in 147 Taiwanese patients with mild-to-moderate essential hypertension (diastolic blood pressure [DBP] 90-109 mmHg). After 6 weeks' treatment, telmisartan produced a significantly greater reduction from baseline in the primary endpoint of trough seated DBP compared with enalapril 10 mg (11.7 vs. 8.7 mmHg, respectively; p = 0.02). Numerically greater reductions compared with baseline in seated systolic blood pressure (SBP), standing DBP, and standing SBP were achieved with telmisartan compared with enalapril. Also, numerically greater proportions of patients achieved blood pressure control (DBP/systolic blood pressure [SBP] <90/140 mmHg) and responded to treatment (reduction from baseline in trough seated DBP > or = 10 mmHg and/or post-treatment DBP <90 mmHg; reduction from baseline in trough seated SBP > or = 10 mmHg and/or post-treatment SBP <140 mmHg) with telmisartan 40 mg compared with enalapril 10 mg. Although both treatments were well tolerated, the incidence of cough was markedly lower with telmisartan 40 mg (8.5%) than with enalapril 10 mg (18.4%) in this population of Taiwanese hypertensive patients.

Gene therapy progress and prospects: recent progress in transgene and RNAi expression cassettes.

Plasmid expression cassette design must include a thoughtful analysis of potentially every nucleotide comprising a covalently closed circular or end-protected linear DNA. This review will discuss recent studies in unraveling the mechanisms of postdelivery gene silencing, codon optimization and promoter identification. The recent discovery of potent RNA interference (RNAi) mechanisms for sequence-specific gene silencing has also invoked a great deal of interest in development of expression cassettes that can produce double-stranded RNA molecules for RNAi. Expression cassettes based on both RNA polymerase II and polymerase III transcription units that generate double-stranded RNA molecules for RNAi will also be discussed.

Secondary electron transfer processes in membranes of Heliobacillus mobilis.

Picosecond transient absorption difference spectroscopic experiments were performed on membranes of the antenna/reaction center complex of Heliobacillus mobilis to study the electron transfer processes. Particular emphasis was placed on the blue spectral region, where the difference spectra of iron-sulfur centers and quinones are significantly different. Spectra were measured at room temperature in the wavelength region from 400 to 470 nm and from 630 to 730 nm. Laser excitation was into the 788 nm Q gamma band of the bacteriochlorophyll g of the reaction center complex. Global analysis in both wavelength regions reveals three kinetic components. A 25 ps phase originates from the decay of the excited state of antenna to form the primary charge-separated state P798+A0-; a 600 ps component is assigned to the electron transfer from the primary electron acceptor A0 to a secondary electron acceptor; a nondecaying component on the time scale measured represents the formation of the secondary charge-separated state. When the secondary electron acceptors were reduced by adding dithionite at pH 11, the 600 ps component disappeared. Only a 25 ps component and a constant were observed in the 630-730 nm region. The 25 ps component is assigned to the excitation decay in the antenna and the formation of P798+A0-, just as in the nonreduced sample. In the reduced sample, the P798+A0- state does not decay on the time scale measured. In the 400-470 nm region, the same kinetic behavior was observed. The absorption difference spectra of the primary and the secondary electron acceptor were constructed from different charge-separated states.(ABSTRACT TRUNCATED AT 250 WORDS)

Simian virus 40 late mRNA leader sequences involved in augmenting mRNA accumulation via multiple mechanisms, including increased polyadenylation efficiency.

We have examined the contribution of 5' leader sequences to expression directed by the simian virus 40 (SV40) late promoter. These studies showed that addition of sequences which contain the late leader 3' splice site to the late promoter led to an increase in the accumulation of mRNA expressed by the promoter. No other sequences within the leader region, between SV40 positions 334 and 560, exhibited a substantial influence on mRNA accumulation. The increase was due, at least in part, to the creation of a spliceable mRNA transcript, since mutation of either the 5' or 3' splice site could attenuate the effect. However, sequences at or near the 3' splice site appeared to play a more important role than did the 5' splice site in bringing about this increase. In many instances, mutation of the 3' splice site also led to the accumulation of extended transcripts, whereas mutation of the 5' splice site did not produce this result in any instance. Analysis of these extended transcripts showed that they retained sequences normally lost upon cleavage and polyadenylation. This finding suggested that mutation of the 3' splice site sequence led to decreases in the efficiency of polyadenylation. We propose that the SV40 late leader sequences positively contribute to expression of the viral late genes by increasing mRNA accumulation via multiple mechanisms, including the enhancement of pre-mRNA polyadenylation efficiency.

Mutations in the herpes simplex virus major DNA-binding protein gene leading to altered sensitivity to DNA polymerase inhibitors.

Five herpes simplex virus mutants containing temperature-sensitive mutations in the gene for the major DNA-binding protein were assayed for their sensitivities to the DNA polymerase inhibitors aphidicolin and phosphonoacetic acid (PAA). Four of the mutants (tsA1, tsA15, tsA24, and tsA42) exhibited altered sensitivity to one or both of the inhibitors relative to the wild-type parent. In tsA1, a mutation or mutations conferring aphidicolin and PAA hypersensitivity were mapped by corescue with the temperature-sensitivity marker of tsA1 to a region of the DNA-binding protein locus, between map coordinates 0.385 and 0.398. The mutation conferring PAA hypersensitivity in tsA24 similarly corescued with the tsA24 temperature-sensitivity marker, mapping to the DNA-binding protein locus between coordinates 0.398 and 0.413. Thus, mutations outside the DNA polymerase locus and within the DNA-binding protein locus can confer altered sensitivity to certain DNA polymerase inhibitors. Assays of the aphidicolin and PAA sensitivities of ts+ recombinants derived by marker rescue of the DNA-binding protein mutants revealed the presence of additional mutations, separable from the ts mutations, in each of three mutants examined. One such mutation, which contributed to the aphidicolin-hypersensitivity phenotype of tsA1, mapped between coordinates 0.422 and 0.448, and resides, most probably, within the DNA polymerase locus. These additional mutations possibly confer compensating modifications to the DNA polymerase such that functional interaction with altered DNA-binding protein is restored. These findings provide strong evidence that the major DNA-binding protein and the DNA polymerase of herpes simplex virus interact in infected cells.

Mutation within the herpes simplex virus DNA polymerase gene conferring resistance to (R)-9-(3,4-dihydroxybutyl)guanine.

Five herpes simplex virus mutants known or presumed to contain mutations in their DNA polymerase genes conferring resistance to acyclovir and arabinosyladenine also proved to exhibit some degree of resistance to (R)-9-(3,4-dihydroxybutyl)guanine (buciclovir). For one mutant, a buciclovir resistance mutation was mapped to a region of the viral DNA polymerase gene proposed to encode the deoxynucleoside 5'-triphosphate binding domain. These data implicate the viral polymerase as a target of buciclovir action that contributes to its antiviral selectivity.

Spectral heterogeneity and time-resolved spectroscopy of excitation energy transfer in membranes of Heliobacillus mobilis at low temperatures.

Transient absorption difference spectra in the Qy absorption band from membranes of Heliobacillus mobilis were recorded at 140 and 20 K upon 200 fs laser pulse excitation at 590 nm. Excitation transfer from short wavelength absorbing forms of bacteriochlorophyll g to long wavelength bacteriochlorophyll g occurred within 1-2 ps at both long wavelength bacteriochlorophyll g occurred within 1-2 ps at both temperatures. In addition, a slower energy transfer process with a time constant of 15 ps was observed at 20 K within the pool of long wavelength-absorbing bacteriochlorophyll g. Energy transfer from long wavelength antenna pigments to the primary electron donor P798 was observed, yielding the primary charge-separated state P798+A0-. The time constant for this process was 30 ps at 140 K and about 70 ps at 20 K. A decay component with smaller amplitude and a lifetime of up to hundreds of picoseconds was observed that was centered around 814 nm at 20 K. Kinetic simulations using simple lattice models reproduce the observed decay kinetics at 295 and 140 K, but not at 20 K. The kinetics of energy redistribution within the spectrally heterogeneous antenna system at low temperature argue against a simple "funnel" model for the organization of the antenna of Heliobacillus mobilis and favor a more random spatial distribution of spectral forms. However, the relatively high rate of energy transfer from long wavelength antenna bacteriochlorophyll g to the primary electron donor P798 at low temperature is difficult to explain with either of these models.

The efficacy of a DNA vaccine encoding herpes simplex virus type 1 (HSV-1) glycoprotein D in decreasing ocular disease severity following corneal HSV-1 challenge.

Antiviral effects of a DNA vaccine against herpes simplex virus 1 (HSV-1) glycoprotein D (gD) were evaluated in eight week-old female BALB/c mice. The nuclease-insensitive construct (gD-ASOR) consisted of an HSV-1 gD encoding plasmid coupled to asialo orosomucoid (ASOR), targeting it to cells bearing ASOR receptors. Mice were immunized on day 0 and 7 with 10 microg doses of gD-ASOR or control substances. Fourteen days later, mice were infected by the corneal route with 10(5) pfu or 10(6) pfu HSV-1, strain 17syn+. Immunized mice showed a significant decrease in ocular disease severity over a 21-day observation period following infection compared to sham-immunized mice. Acute replication kinetic assays demonstrated a 100-fold decrease in viral titers on day 6 in trigeminal ganglia from immunized BALB/c mice compared to sham-immunized mice. Immunized mice showed a significant increase in numbers of CD4(+)T cells infiltrating the trigeminal ganglia at day 6 post infection compared to sham-immunized mice. Significant differences were not seen in latent viral reservoir between immunized and unimmunized mouse groups. Immunization with gD-ASOR decreased the severity of acute ocular HSV-1 infection, induced a CD4(+) T cell response, decreased the viral load in the trigeminal ganglia, but did not diminish viral latency.

Time-resolved spectroscopy of energy and electron transfer processes in the photosynthetic bacterium Heliobacillus mobilis.

The kinetics of excitation energy transfer and electron transfer processes within the membrane of Heliobacillus mobilis were investigated using femtosecond transient absorption difference spectroscopy at room temperature. The kinetics in the 725- to 865-nm region, upon excitation at 590 and 670 nm, were fit using global analysis. The fits returned three kinetic components with lifetimes of 1-2 ps and 27-30 ps, and a component that does not decay within several nanoseconds. The 1- to 2-ps component is attributed to excitation equilibration to form a thermally relaxed excited state. The 27- to 30-ps phase corresponds to the decay of the relaxed excited state to form a charge-separated state. The intrinsic energy and electron transfer rates were estimated using the experimental results and theoretical models for excitation migration and trapping dynamics. Taking into account the number of antenna pigments and their spectral distribution, an upper limit of 1.2 ps for the intrinsic time constant for charge separation in the reaction center is calculated. This upper limit corresponds with the trapping-limited case for excitation migration and trapping. Reduction of the primary electron acceptor A0 was observed in the 640 to 700 nm region using excitation at 780 nm. An instantaneous absorbance increase followed by a decay of about 30 ps was observed over a broad wavelength region due to the excited state absorption and decay of BChl g molecules in the antenna. In addition, a narrow bleaching band centered at 670 nm grows in with an apparent time constant of about 1.0 ps, superimposed on the 30-ps absorbance increase due to excited state absorption. Measurements on a longer time scale showed that besides the 670 nm pigment a BChl g molecule absorbing near 785 nm may be involved in the primary charge separation, and that this pigment may be in equilibrium with the 670 nm pigment. The bleaching bands at 670 nm and 785nm recovered with a time constant of about 600 ps, due to forward electron transport to a secondary electron acceptor. Energy and electron transfer properties of H. mobilis membranes are compared with Photosystem 1, to which the heliobacteria bear an evolutionary relationship.

Spectroscopic evidence for the presence of an iron-sulfur center similar to Fx of Photosystem I in Heliobacillus mobilis.

Treatment of membranes of Heliobacillus mobilis with high concentrations of the chaotropic agent urea resulted in the removal of the iron-sulfur centers FA and FB from the reaction center, as indicated by EPR spectra under strongly reducing conditions. In urea-treated membranes, transient absorption measurements upon a laser flash indicated a recombination between the photo-oxidized primary donor P798+ and a reduced acceptor with a time constant of 20 ms at room temperature. Benzylviologen, vitamin K-3 and methylene blue were found to accept electrons from the reduced acceptor efficiently. A differential extinction coefficient of 225-240 mM-1 cm-1 at 798 nm was determined from experiments in the presence of methylene blue. Transient absorption difference spectra between 400 and 500 nm in the presence and absence of artificial acceptors indicated that the electron acceptor involved in the 20 ms recombination has an absorption spectrum similar to that of an iron-sulfur center. This iron-sulfur center was assigned to be analogous to Fx of Photosystem I. Our results provide evidence in support of the presence of Fx in heliobacteria, which was proposed on the basis of the reaction center polypeptide sequence (Liebl et al. (1993) Proc. Natl. Acad. Sci. USA 90: 7124-7128). Implications for the electron transfer pathway in the reaction center of heliobacteria are discussed.

Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition.

Herpes simplex virus (HSV) encodes a DNA polymerase that is similar in several respects to the replicative mammalian DNA polymerase alpha. Recently, these and other DNA polymerases have been shown to share several regions of protein sequence similarity. Despite these similarities, antiviral drugs that mimic natural polymerase substrates specifically inhibit herpesvirus DNA polymerases. To study amino acids involved in substrate and drug recognition, we have characterized and mapped altered drug sensitivity markers of nine HSV pol mutants and sequenced the relevant portions of these mutants. The mutations were found to occur within four relatively small regions. One such region, which we designate region A, has sequence similarity only to DNA polymerases that are sensitive to certain antiviral drugs. The other three regions contain sequences that are similar among various DNA polymerases. The multiple mutations occurring within two of these regions make it likely that the regions interact directly with drugs and substrates. Our results lead us to favor a model in which protein folding allows interactions among the four regions to form the substrate and drug binding sites.

Murine response to DNA encoding herpes simplex virus type-1 glycoprotein D targeted to the liver.

Plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) was complexed with asialoorosomucoid conjugated to poly-L-lysine. Following its intravenous injection into BALB/c mice, this complex was targeted to the liver. Liver cells expressing gD-1 were detected immunohistochemically through day 6 post-immunization, while gD-1 DNA was detectable through 14 days post-immunization. Decline of gD-1 expression and detectable gD-1 DNA in the liver correlated with influx of T cells, predominantly CD4(+). The ASOR-poly-L-lysine DNA carrier system promotes hepatic expression of gD-1 and may be useful in vaccination against herpes simplex virus type-1.