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PubMed2003

Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatography.

A hydrophobic interaction HPLC method was developed for the quantification of plasmid DNA and assessment of its purity in crude Escherichia coli lysates and other process streams. A Phenyl Sepharose Source (Amersham Biosciences) column was used to separate the double-stranded plasmid DNA molecules from the more hydrophobic impurities present in the process streams. The method is rapid (each analysis takes 7 min), reproducible, easy to perform and does not require previous digestion of RNA in samples with RNase or other pre-treatment. Furthermore, it is capable of handling heavily contaminated samples, with less than 5% of plasmid DNA thus constituting a good alternative to other less robust analytical techniques currently in use.

Calibration↗
PubMed2005

Cost-effectiveness inferences from bootstrap quadrant confidence levels: three degrees of dominance.

When with at least 95% confidence a new treatment is shown to be not only less costly (LC), but also more effective (ME), than a current treatment, that new treatment can be said to "strictly dominate" the current treatment statistically. But what can be said when head-to-head treatment comparisons turn out to be less clear-cut than this? Here, we propose two additional sets of specific LC and/or ME confidence thresholds to define the concepts of "some dominance" and "much dominance." Confidence levels associated with entire quadrants of the incremental cost-effectiveness (ICE) plane are easily computed using the same bootstrapping techniques used to estimate an "acceptability curve." Our two proposed additional "degrees" of dominance, although less stringent than strict dominance, are nevertheless more stringent than commonly accepted approaches using ICE ratio or net benefit calculations. To illustrate analysis concepts, we use data from a randomized, double-blind, placebo- and active comparator-controlled clinical registration trial for treatment of major depressive disorder (MDD). As is typical, our case study is rather small and short term, providing outcome information for a total of only 264 patients during their initial 8 weeks of acute-phase MDD treatment. Thus, we focus attention on sensitivity analyses, showing that the bootstrap distribution of cost-effectiveness uncertainty is robust across two alternative ways of measuring overall effectiveness and three alternative ways of imputing missing values. Evaluation of the balance between cost and benefit is particularly difficult when a new pharmacological treatment is first introduced, yet information of this sort is highly desired by decision makers. We show that, even with only a relatively modest amount of clinical trial information, sensitivity analyses can still confirm that cost-effectiveness comparisons are being made in a consistent fashion. In contrast, extensive follow-up comparisons using data from actual clinical practice will almost always ultimately be needed to better inform health policy makers.

Adult↗
PubMed1999

Conversion of C57Bl/6 mice from a tumor promotion-resistant to a -sensitive phenotype by enhanced ornithine decarboxylase expression.

A transgenic mouse model was developed in which ornithine decarboxylase (ODC) can be overexpressed in a tissue-specific and regulated manner. Hair follicle keratinocytes were targeted by use of a bovine keratin 6 (K6) promoter/regulatory region, and regulation was accomplished by using the tetracycline-regulated transactivator/tetracycline-response element system. Double-transgenic mice carrying both transgenes (K6/tetracycline-regulatable transactivator protein (tTA) and tetracycline-response element/Odc) on a C57Bl/6 background had no obvious phenotypic abnormalities in the absence (Odc transgene-expressed) of doxycycline (a tetracycline analog) in the drinking water. However, induction of K6-driven tTA expression by the tumor promoter (12-O-tetradecanoylphorbol-13-acetate) (TPA) led to very high levels of epidermal ODC activity and robust hyperplasia, especially involving hair follicles. Both effects were abolished by inclusion of doxycycline in the drinking water to repress transgene expression. Finally, the number of papillomas that developed in a standard (7,12-dimethybenz[a]anthracene) (DMBA)/TPA protocol was greatly reduced in mice in which transgenic Odc expression was repressed by doxycycline. Our results demonstrated that the higher levels of ODC expression produced in the transgenic model in the induced versus the repressed condition make the normally promotion-resistant C57Bl/6 strain much more sensitive to the short-term and long-term (i.e., tumor-promoting) effects of TPA.

Animals↗
PubMed2006

An integrated analysis of five double-blind, randomized controlled trials evaluating the safety and efficacy of a hyaluronan product for intra-articular injection in osteoarthritis of the knee.

OBJECTIVE: Five double-blind, randomized, saline-controlled trials (RCTs) were included in the United States marketing application for an intra-articular hyaluronan (IA-HA) product for the treatment of osteoarthritis (OA) of the knee. We report an integrated analysis of the primary Case Report Form (CRF) data from these trials. METHOD: Trials were similar in design, patient population and outcome measures - all included the Lequèsne Algofunctional Index (LI), a validated composite index of pain and function, evaluating treatment over 3 months. Individual patient data were pooled; a repeated measures analysis of covariance was performed in the intent-to-treat (ITT) population. Analyses utilized both fixed and random effects models. Safety data from the five RCTs were summarized. RESULTS: A total of 1155 patients with radiologically confirmed knee OA were enrolled: 619 received three or five IA-HA injections; 536 received "placebo" saline injections. In the active and control groups, mean ages were 61.8 and 61.4 years; 62.4% and 58.8% were women; baseline total Lequèsne scores 11.03 and 11.30, respectively. Integrated analysis of the pooled data set found a statistically significant reduction (P < 0.001) in total Lequèsne score with hyaluronan (HA) (-2.68) vs placebo (-2.00); estimated difference -0.68 (95% CI: -0.56 to -0.79), effect size 0.20. Additional modeling approaches confirmed robustness of the analyses. CONCLUSIONS: This integrated analysis demonstrates that multiple design factors influence the results of RCTs assessing efficacy of intra-articular (IA) therapies, and that integrated analyses based on primary data differ from meta-analyses using transformed data.

Aged↗
PubMed1994

Fluoxetine attenuates alcohol intake and desire to drink.

Several serotonin uptake inhibitors, including the long-acting fluoxetine, have been found to decrease alcohol intake in moderately dependent alcoholics. While the mechanism of their effect is not fully elucidated, a previous study with citalopram indicated that decreased desire to drink may be an important factor. Therefore, we tested fluoxetine effects on alcohol intake and desire to drink in a placebo-controlled study. Subjects, recruited by advertisement, were mildly/moderately dependent alcoholics (12 male, four female, aged 19-59 years, healthy, non-depressed) who did not believe they had a drinking problem and were not requesting treatment. After a 1 week baseline they received, single-blind, 2 weeks placebo followed by 2 weeks fluoxetine 60 mg/day. As out-patients, subjects recorded daily standard drinks (13.6 g ethanol) and rated interest, desire, craving and liking for alcohol biweekly. Each out-patient period was immediately followed by a double-blind experimental drinking session. Out-patient daily drinks slightly decreased during fluoxetine to 6.6 +/- 0.9 (mean +/- S.E.M.) compared with during placebo (7.16 +/- 0.95, p = 0.07, N.S.) and baseline (7.18 +/- 1.0, p > 0.1, N.S.). Desire, interest and craving for alcohol decreased during fluoxetine vs placebo baseline (p < 0.05), but not vs placebo. Appetite loss and decrease in food intake (p < 0.01, fluoxetine vs placebo) correlated with each other (r = 0.91, p < 0.01) but neither correlated with decrease in alcohol intake (appetite: r = 0.26, N.S.; food intake: r = 0.22, N.S.). Weight loss occurred during fluoxetine (p < 0.05 vs placebo) but did not correlate with decrease in alcohol intake (r = 0.1, N.S.). In the experimental drinking sessions after placebo and fluoxetine treatments subjects rated their desire for each of 18 mini-drinks (each one-third of a standard drink) offered at 5 min intervals. Fluoxetine decreased desire to drink throughout the sessions; both mean and maximum desire ratings were lower after fluoxetine than after placebo (ANOVA, p < 0.05). Therefore, fluoxetine seems to have a robust effect on decreasing desire for alcohol. We propose that in the absence of intention by subjects to reduce drinking, their habitual drinking patterns mitigated against reduced consumption in the out-patient phase. However, fluoxetine could be a useful adjunct for patients in a treatment context who are motivated to reduce their drinking.

Adult↗
PubMed2006

Double-blind evaluation and benchmarking of survival models in a multi-centre study.

Accurate modelling of time-to-event data is of particular importance for both exploratory and predictive analysis in cancer, and can have a direct impact on clinical care. This study presents a detailed double-blind evaluation of the accuracy in out-of-sample prediction of mortality from two generic non-linear models, using artificial neural networks benchmarked against a partial logistic spline, log-normal and COX regression models. A data set containing 2880 samples was shared over the Internet using a purpose-built secure environment called GEOCONDA (www.geoconda.com). The evaluation was carried out in three parts. The first was a comparison between the predicted survival estimates for each of the four survival groups defined by the TNM staging system, against the empirical estimates derived by the Kaplan-Meier method. The second approach focused on the accurate prediction of survival over time, quantified with the time dependent C index (C(td)). Finally, calibration plots were obtained over the range of follow-up and tested using a generalization of the Hosmer-Lemeshow test. All models showed satisfactory performance, with values of C(td) of about 0.7. None of the models showed a systematic tendency towards over/under estimation of the observed survival at tau=3 and 5 years. At tau=10 years, all models underestimated the observed survival, except for COX regression which returned an overestimate. The study presents a robust and unbiased benchmarking methodology using a bespoke web facility. It was concluded that powerful, recent flexible modelling algorithms show a comparative predictive performance to that of more established methods from the medical and biological literature, for the reference data set.

Benchmarking↗
PubMed2006

A randomized trial of an N-methyl-D-aspartate antagonist in treatment-resistant major depression.

CONTEXT: Existing therapies for major depression have a lag of onset of action of several weeks, resulting in considerable morbidity. Exploring pharmacological strategies that have rapid onset of antidepressant effects within a few days and that are sustained would have an enormous impact on patient care. Converging lines of evidence suggest the role of the glutamatergic system in the pathophysiology and treatment of mood disorders. OBJECTIVE: To determine whether a rapid antidepressant effect can be achieved with an antagonist at the N-methyl-D-aspartate receptor in subjects with major depression. DESIGN: A randomized, placebo-controlled, double-blind crossover study from November 2004 to September 2005. SETTING: Mood Disorders Research Unit at the National Institute of Mental Health. Patients Eighteen subjects with DSM-IV major depression (treatment resistant). INTERVENTIONS: After a 2-week drug-free period, subjects were given an intravenous infusion of either ketamine hydrochloride (0.5 mg/kg) or placebo on 2 test days, a week apart. Subjects were rated at baseline and at 40, 80, 110, and 230 minutes and 1, 2, 3, and 7 days postinfusion. Main Outcome Measure Changes in scores on the primary efficacy measure, the 21-item Hamilton Depression Rating Scale. RESULTS: Subjects receiving ketamine showed significant improvement in depression compared with subjects receiving placebo within 110 minutes after injection, which remained significant throughout the following week. The effect size for the drug difference was very large (d = 1.46 [95% confidence interval, 0.91-2.01]) after 24 hours and moderate to large (d = 0.68 [95% confidence interval, 0.13-1.23]) after 1 week. Of the 17 subjects treated with ketamine, 71% met response and 29% met remission criteria the day following ketamine infusion. Thirty-five percent of subjects maintained response for at least 1 week. CONCLUSIONS: Robust and rapid antidepressant effects resulted from a single intravenous dose of an N-methyl-D-aspartate antagonist; onset occurred within 2 hours postinfusion and continued to remain significant for 1 week.

Adult↗
PubMed2002

An economic analysis of amiodarone versus placebo for the prevention of atrial fibrillation after open heart surgery.

STUDY OBJECTIVE: To determine if the additional costs of oral amiodarone in patients undergoing open heart surgery would be offset by reductions in the frequency of atrial fibrillation. DESIGN: Piggyback cost analysis of the data from a randomized, double-blind, placebo-controlled trial. SETTING: Urban academic hospital. PATIENTS: Two hundred twenty elderly patients (> or = 60 yrs old) undergoing open heart surgery. INTERVENTION: Hospital costs of open heart surgery in patients given amiodarone for the prevention of atrial fibrillation and in prespecified subgroups were compared with those for patients given placebo (i.e., standard care with beta-blockers alone). MEASUREMENTS AND MAIN RESULTS: Total hospital costs incurred were $15,565 +/- $9832 and $16,126 +/- $8043 in the amiodarone and placebo groups, respectively (p=0.12). General ward, intensive care unit, operating room, pharmacy, and costs in all other departments were similar between the groups (p>0.05 for all comparisons). Because costs were similar but amiodarone was more effective than placebo, amiodarone was cost-effective compared with placebo. Amiodarone remained cost-effective compared with placebo regardless of the following subgroup characteristics: rapid or slow loading strategy, no history of atrial fibrillation or heart failure, age older than 70 years, and no tolerance to preoperative beta-blockers. Moreover, in the one-way sensitivity analysis, the findings remained robust to changes in effectiveness and cost of amiodarone. CONCLUSION: Routine prophylaxis with amiodarone is cost-effective compared with placebo. Future studies should examine the cost-effectiveness of selective prophylaxis, and primary cost-effectiveness studies should be conducted to validate these findings.

Aged↗
PubMed2001

Efficacy of a mixed amphetamine salts compound in adults with attention-deficit/hyperactivity disorder.

BACKGROUND: We report on a controlled trial of a mixed amphetamine salts compound (Adderall, dextroamphetamine sulfate, dextro-, levoamphetamine sulfate, dextroamphetamine aspartate, levoamphetamine aspartate, and dextroamphetamine saccharate) in the treatment of adult attention-deficit/hyperactivity disorder (ADHD). METHODS: This was a 7-week, randomized, double-blind, placebo-controlled, crossover study of Adderall in 27 well-characterized adults satisfying full DSM-IV criteria for ADHD of childhood onset and persistent symptoms into adulthood. Medication was titrated up to 30 mg twice a day. Outcome measures included the ADHD Rating Scale and the Clinical Global Impression Score. Comorbid psychiatric disorders were assessed to test for potential effects on treatment outcome. RESULTS: Treatment with Adderall at an average oral dose of 54 mg (administered in 2 daily doses) was effective and well tolerated. Drug-specific improvement in ADHD symptoms was highly significant overall (42% decrease on the ADHD Rating Scale, P<.001), and sufficiently robust to be detectable in a parallel groups comparison restricted to the first 3 weeks of the protocol (P<.001). The percentage of subjects who improved (reduction in the ADHD rating scale of > or =30%) was significantly higher with Adderall treatment than with a placebo (70% vs 7%; P =.001). CONCLUSIONS: Adderall was effective and well tolerated in the short-term treatment of adults with ADHD. More work is needed to evaluate the long-term effects of Adderall, or other amphetamine compounds, in the treatment of adults with ADHD.

Adult↗
PubMed1995

Fos expression induced by changes in arterial pressure is localized in distinct, longitudinally organized columns of neurons in the rat midbrain periaqueductal gray.

The distribution of neurons expressing Fos within the periaqueductal gray (PAG) following pharmacologically induced high or low blood pressure was examined to determine (1) if PAG neurons are responsive to changes in arterial pressure (AP) and (2) the relationship of these cells to the functionally defined hypertensive and hypotensive columns in PAG. Changes in AP differentially induced robust Fos expression in neurons confined to discrete, longitudinally organized columns within PAG. Increased AP produced extensive Fos-like immunoreactivity within the lateral PAG, beginning at the level of the oculomotor nucleus. At the level of the dorsal raphe, Fos expression induced by increased AP shifted dorsally, into the dorsolateral division of PAG; this pattern of Fos labeling was maintained throughout the caudal one-third of PAG. Double-labeling for Fos and nicotinamide adenine dinucleotide phosphate diaphorase confirmed that Fos-positive cells induced by increased AP were located in the dorsolateral division of PAG at these caudal levels. Fos positive cells were codistributed, but not colocalized, with nicotinamide adenine dinucleotide phosphate diaphorase-positive cells. Decreased AP evoked a completely different pattern of Fos expression. Fos-positive cells were predominantly located within the ventrolateral PAG region, extending from the level of the trochlear nucleus through the level of the caudal dorsal raphe. Double-labeling studies for Fos and serotonin indicated that only 1-2 double-labeled cells per section were present. Saline infusion resulted in very few Fos-like immunoreactive cells, indicating that volume receptor activation does not account for Fos expression in PAG evoked by changes in AP. These results indicate that (1) substantial numbers of PAG neurons are excited by pharmacologically induced changes in AP and (2) excitatory barosensitive PAG neurons are anatomically segregated based on their responsiveness to a specific directional change in AP.

Animals↗
PubMed1977

Some population genetic models combining artificial and natural selection pressures in the presence of assortative mating.

We have attempted quantitatively through a series of assortative mating models to gain insight into the interaction between the usually antagonistic tendencies of artifical and natural selection pressures. We summarize some of the robust conclusions. In cases where natural selection is expressed only through the phenotype and acts in the opposite direction to the culling incline, then fixation of the dominant or recessive type can be achieved and which occurs depends critically on the initial composition of the population and the magnitude of the degree of culling compared to the selection coefficients. With traits determined at two loci in the case that the double heterozygote is the desired kind, the effect of selfing can only be overcome by very strong artificial selection pressures (high culling order). The degree of culling to achieve its objective can be relaxed with weakening of linkage. The relevant comparison is r2+(1-r)2less than 2(1-c) indicating the precise extent of culling needed, to prevent fixation. The relationships are more complex when natural selection forces are also involved (see Model IV).

Alleles↗
PubMed2002

The effects of sub-chronic administration of hydrocortisone on hormonal and psychological responses to L-tryptophan in normal male volunteers.

RATIONALE: 5-Hydroxytryptamine(1A) (5-HT(1A)) receptor function has been shown to be attenuated by corticosteroid hormones in a variety of animal experimental paradigms. It has been suggested that this effect may be central to the pathophysiology of severe depressive illness in humans, a condition in which 5-HT(1A) receptor function is reduced and corticosteroid hormone levels are elevated. Evidence suggests that the hormonal response to L-tryptophan ( L-TRP) is mediated by 5-HT(1A) receptors. This response has been shown to be reduced following acute administration of hydrocortisone, and we hypothesised that sub-chronic administration of hydrocortisone would also blunt it. OBJECTIVES: To examine the effects of sub-chronic administration of hydrocortisone on hormonal and psychological responses to L-TRP infusion in healthy male subjects. To ascertain whether cortisol was exerting effects on prolactin release directly at the pituitary rather than via hypothalamic 5-HT(1A) receptors, a thyroid-releasing hormone (TRH) challenge test was performed. METHODS: Fourteen healthy male volunteers took part in a random-order, double-blind, placebo-controlled study, in which 20 mg hydrocortisone or placebo was administered twice daily for 7 days before infusion of L-TRP. A TRH challenge was administered to eight of the subjects following the L-TRP infusion. RESULTS: Pre-treatment with hydrocortisone significantly reduced the growth hormone (GH) and cortisol responses, but not the prolactin (PRL) response to the infusion. TRH administration caused a robust increase in PRL, but this response was not attenuated by hydrocortisone pre-treatment. The TSH response to TRH was blunted. There was no effect of pre-treatment on psychological responses to L-TRP. CONCLUSIONS: The attenuation in GH response following hydrocortisone pre-treatment could indicate a reduction in 5-HT(1A) receptor function, although it is probable that it is attributable to the action of hydrocortisone at the pituitary level. More precise, non-neuroendocrine models of 5-HT(1A) receptor function are necessary to clarify this.

Adult↗
PubMed2005

Method for cortical bone structural analysis from magnetic resonance images.

RATIONALE AND OBJECTIVES: Quantitative evaluation of cortical bone architecture as a means to assess bone strength typically is accomplished on the basis of images obtained by means of dual-energy X-ray absorptiometry (DXA) or computed tomography. Magnetic resonance (MR) imaging has potential advantages for this task in that it allows imaging in arbitrary scan planes at high spatial resolution. However, several hurdles have to be overcome to make this approach practical, including resolution of issues related to nonlinear receive coil sensitivity, variations in marrow composition, and the presence of periosteal isointense tissues, which all complicate segmentation. The aim of this study is to develop MR acquisition and analysis methods optimized for the detection of cortical boundaries in such complex geometries as the femoral neck. MATERIALS AND METHODS: Cortical boundary detection is achieved by radially tracing intensity profiles that intersect the periosteal and endosteal boundaries of bone. Profiles subsequently are normalized to the intensity of the marrow signal, processed with morphologic image operators, and binarized. The resulting boundaries are mapped back onto the spatial image, and erroneous boundary points are removed. From the detected cortical boundaries, cortical cross-sectional area and thickness are computed. The method was evaluated on cortical bone specimens and human volunteers on the basis of high-resolution images acquired at a 1.5-Tesla field strength. To assess whether the method is sensitive to detect the expected dependencies of cortical parameters in weight-bearing bone on overall habitus, 10 women aged 46-73 years (mean age, 56 years) underwent the cortical imaging protocol in the proximal femur, and results were compared with DXA bone mineral density parameters of the hip and spine. RESULTS: Reproducibility was approximately 2%. Double oblique images of the femoral neck in the 10 women studied showed that cortical cross-sectional area correlated strongly with height (r = 0.88; p = .0008), whereas cortical diameter versus age approached significance (r = 0.61; p = .06). Measurements in specimens of some cortical parameters indicated resolution dependence. However, note that specimen ranking within each parameter remained constant across all resolutions studied. CONCLUSION: Data suggest the new method to be robust and applicable on standard clinical MR scanners at arbitrary anatomic locations to yield clinically meaningful quantitative results.

Absorptiometry, Photon↗
PubMed1997

Phylogeography and host association in a pollinating seed parasite Greya politella (Lepidoptera: Prodoxidae).

Inferring the historical context of ecological diversification is an important step in understanding the way that population-level processes result in a diversity of species and interactions in communities. We performed a phylogeographic analysis of mitochondrial DNA haplotypes from the pollinating seed parasite Greya politella (Lepidoptera: Prodoxidae) in order to determine the degree to which populations were structured according to geographical location and host-plant association. Ninety-eight individuals were sampled from 29 locations ranging from southern California to western Idaho. Restriction-site variation in 87 individuals (27 populations) was screened by digestion with 11 endonucleases, followed by Southern blotting; 38 restriction-site positions were mapped by double digests. Haplotypes were further defined by generating fragments 251 bases in length via PCR, screening them for sequence variation using denaturing gel gradient electrophoresis (DGGE), and sequencing the resulting variants. Parsimony analysis of the resulting 12 restriction-site and 15 sequence haplotypes indicated strong geographical structuring of populations: (i) most populations were monomorphic for haplotype; (ii) haplotypes from California and the Pacific Northwest (Oregon, Washington and Idaho) formed robust monophyletic groups. Population structure was significant both within and between the two regions, as reflected by NST. Patterns of host-plant association and haplotype phylogeny suggest that populations have recently undergone host-plant shifts in many different parts of the species range, although the direction and number of host shifts cannot be determined at the present level of sampling resolution.

Animals↗
PubMed2003

Single-stranded DNA-binding proteins PURalpha and PURbeta bind to a purine-rich negative regulatory element of the alpha-myosin heavy chain gene and control transcriptional and translational regulation of the gene expression. Implications in the repression of alpha-myosin heavy chain during heart failure.

The alpha-myosin heavy chain is a principal molecule of the thick filament of the sarcomere, expressed primarily in cardiac myocytes. The mechanism for its cardiac-restricted expression is not yet fully understood. We previously identified a purine-rich negative regulatory (PNR) element in the first intron of the gene, which is essential for its cardiac-specific expression (Gupta, M., Zak, R., Libermann, T. A., and Gupta, M. P. (1998) Mol. Cell. Biol. 18, 7243-7258). In this study we cloned and characterized muscle and non-muscle factors that bind to this element. We show that two single-stranded DNA-binding proteins of the PUR family, PURalpha and PURbeta, which are derived from cardiac myocytes, bind to the plus strand of the PNR element. In functional assays, PURalpha and PURbeta repressed alpha-myosin heavy chain (alpha-MHC) gene expression in the presence of upstream regulatory sequences of the gene. However, from HeLa cells an Ets family of protein, Ets-related protein (ERP), binds to double-stranded PNR element. The ERP.PNR complex inhibited the activity of the basal transcription complex from homologous as well as heterologous promoters in a PNR position-independent manner, suggesting that ERP acts as a silencer of alpha-MHC gene expression in non-muscle cells. We also show that PUR proteins are capable of binding to alpha-MHC mRNA and attenuate its translational efficiency. Furthermore, we show robust expression of PUR proteins in failing hearts where alpha-MHC mRNA levels are suppressed. Together, these results reveal that (i) PUR proteins participate in transcriptional as well as translational regulation of alpha-MHC expression in cardiac myocytes and (ii) ERP may be involved in cardiac-restricted expression of the alpha-MHC gene by preventing its expression in non-muscle cells.

Animals↗
PubMed1998

Effects of tryptophan depletion vs catecholamine depletion in patients with seasonal affective disorder in remission with light therapy.

BACKGROUND: Although hypotheses about the therapeutic mechanism of action of light therapy have focused on serotonergic mechanisms, the potential role, if any, of catecholaminergic pathways has not been fully explored. METHODS: Sixteen patients with seasonal affective disorder who had responded to a standard regimen of daily 10000-lux light therapy were enrolled in a double-blind, placebo-controlled, randomized crossover study. We compared the effects of tryptophan depletion with catecholamine depletion and sham depletion. Ingestion of a tryptophan-free amino acid beverage plus amino acid capsules was used to deplete tryptophan. Administration of the tyrosine hydroxylase inhibitor alpha-methyl-paratyrosine was used to deplete catecholamines. Diphenhydramine hydrochloride was used as an active placebo during sham depletion. The effects of these interventions were evaluated with measures of depression, plasma tryptophan levels, and plasma catecholamine metabolites. RESULTS: Tryptophan depletion significantly decreased plasma total and free tryptophan levels. Catecholamine depletion significantly decreased plasma 3-methoxy-4-hydroxyphenylethyleneglycol and homovanillic acid levels. Both tryptophan depletion and catecholamine depletion, compared with sham depletion, induced a robust increase (P<.001, repeated-measures analysis of variance) in depressive symptoms as measured with the Hamilton Depression Rating Scale, Seasonal Affective Disorder Version. CONCLUSIONS: The beneficial effects of light therapy in the treatment of seasonal affective disorder are reversed by both tryptophan depletion and catecholamine depletion. These findings confirm previous work showing that serotonin plays an important role in the mechanism of action of light therapy and provide new evidence that brain catecholaminergic systems may also be involved.

Adult↗
PubMed2003

Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats.

Plant virus-based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology-dependent RNA degradation mechanism. This phenomenon, called virus-induced gene silencing (VIGS), has potential as a powerful reverse-genetics tool in functional genomic programmes through transient, loss-of-function screens. Here, we describe a method to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production, a critical step in the VIGS response. Incorporation of 40-60 base direct inverted-repeats into a plant viral vector generates RNA molecules that form dsRNA hairpins. A tobacco mosaic virus (TMV)-based vector carrying such inverted-repeats, homologous to a green fluorescent protein (gfp) transgene or an endogenous phytoene desaturase (pds) gene, generated a stronger and more pervasive VIGS phenotype than constructs carrying corresponding cDNA fragments in sense or antisense orientation. Real-time RT-PCR indicated that there was up to a threefold reduction in target mRNA accumulation in the tissues where VIGS was triggered by constructs carrying inverted-repeats compared to those where it was triggered by sense or antisense constructs. Moreover, an enhanced VIGS pds phenotype was observed using a different vector, based on barley stripe mosaic virus, in the monocotyledonous host barley. This demonstrates that VIGS can be significantly improved through the inclusion of small inverted-repeats in plant virus-based vectors, generating a more robust loss-of-function phenotype. This suggests that dsRNA formation can be a limiting factor in the VIGS phenomenon.

Base Sequence↗
PubMed2005

Gender differences in growth hormone response to exercise before and after rhGH administration and the effect of rhGH on the hormone profile of fit normal adults.

OBJECTIVES: Exercise is a potent physiological stimulus of GH secretion. We hypothesized that exogenous recombinant human growth hormone (rhGH) administration through an increase in GH and IGF-I levels would blunt the GH response to exercise. The aim of the study was to examine and compare the impact of rhGH on the exercise-induced GH response in healthy normal men and women. DESIGN AND MEASUREMENTS: Sixty-nine subjects (36 men, 33 women) were randomized to receive low-dose rhGH (0.1 U/kg/day), high dose rhGH (0.2 U/kg/day), or placebo. Subjects were matched for age (24 +/- 3.1), and body mass index (BMI). rhGH was given as a single subcutaneous (s.c.) injection for the first 28 days. All subjects exercised to exhaustion (maximal oxygen consumption--VO2max) before rhGH treatment (Test 1), and on day 28 (Test 2). GH was measured before exercise (time 0), immediately after exercise (time 0') and at 15, 30, 60, 90 and 120 min postexercise. Baseline IGF-I levels were measured before exercise on days 0 and 28. RESULTS: Baseline IGF-I levels showed no gender differences (42.3 women vs. 38.8 nmol/l men) but basal GH values were higher in women (9.9 vs. 1.8 mU/l, P < 0.001). The areas under the GH response curve, for Test 1 were similar in men and women. Peak GH values were higher in women than men (37.9 vs. 23.5 mU/l, but this did not quite reach statistical significance (P = 0.055). In men, administration of rhGH resulted in a significant increase in IGF-I levels over the basal state in both the LD and HD groups (P < 0.0001). In women, the increase in lGF-I levels reached significance only in the HD group (P < 0.0001). On day 28, GH secretion in response to exercise was calculated from the areas under the GH response curve correcting for an exogenous rhGH component (delta AUC). In men, the delta AUC, for Test 2 were similar in all three groups. In women, the delta AUC was higher in the placebo group, than in the HD group (P < 0.02). Free T4 levels decreased significantly in men, and free T3 increased in both men and women, in HD group after the rhGH administration. TSH levels were suppressed only in women. No changes in sex hormones were found in men or women in any of the treatment groups. Conclusions In terms of IGF-I, men are more responsive to rhGH treatment than women. In addition, as men, but not women, were able to overcome the negative feedback control of the elevated IGF-I levels, it seems that exercise may be a more robust stimulus to GH release in men compared to women.

Adult↗