Natural variation in light sensitivity of Arabidopsis.

Because plants depend on light for growth, their development and physiology must suit the particular light environment. Plants native to different environments show heritable, apparently adaptive, changes in their response to light. As a first step in unraveling the genetic and molecular basis of these naturally occurring differences, we have characterized intraspecific variation in a light-dependent developmental process-seedling emergence. We examined 141 Arabidopsis thaliana accessions for their response to four light conditions, two hormone conditions and darkness. There was significant variation in all conditions, confirming that Arabidopsis is a rich source of natural genetic diversity. Hierarchical clustering revealed that some accessions had response patterns similar to known photoreceptor mutants, suggesting changes in specific signaling pathways. We found that the unusual far-red response of the Lm-2 accession is due to a single amino-acid change in the phytochrome A (PHYA) protein. This change stabilizes the light-labile PHYA protein in light and causes a 100-fold shift in the threshold for far-red light sensitivity. Purified recombinant Lm-2 PHYA also shows subtle photochemical differences and has a reduced capacity for autophosphorylation. These biochemical changes contrast with previously characterized natural alleles in loci controlling plant development, which result in altered gene expression or loss of gene function.

Genetic bias in immune responses to a cassette shared by different microorganisms in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease associated with HLA-DRbeta1 alleles which contain the QKRAA amino acid sequence in their third hypervariable region(s). The QKRAA sequence is also expressed by several human pathogens. We have shown previously that an Escherichia coli peptide encompassing QKRAA is a target of immune responses in RA patients. Here we address two questions: first, whether QKRAA may function as an "immunological cassette" with similar, RA-associated, immunogenic properties when expressed by other common human pathogens; and second, what is the influence of genetic background in the generation of these responses. We find that early RA patients have enhanced humoral and cellular immune responses to Epstein-Barr virus and Brucella ovis and Lactobacillus lactis antigens which contain the QKRAA sequence. These results suggest that the QKRAA sequence is an antigenic epitope on several different microbial proteins, and that RA patients recognize the immunological cassette on different backgrounds. ANOVA of immune responses to "shared epitope" antigens in monozygotic twin couples shows that, despite significantly elevated responses in affected individuals, a similarity between pairs is retained, thus suggesting a role played either by hereditary or shared environmental factors in the genesis or maintenance of these responses.

Human fibroblast and human bone marrow cell response to lithographically nanopatterned adhesive domains on protein rejecting substrates.

The separate influence of topographical and chemical cues on cell attachment and spreading are well documented; however, that of duel-cue substrates is less so. In this study graft copolymers that sterically stabilize biological surfaces were employed alongside nanotopographical features fabricated by colloidal lithography. This resulted in the production of a range of substrates whereby the effect of chemistry and or topography on both on human fibroblast and bone marrow cell adhesion and spreading could be observed. The current studies indicate an enhancement of cell response as a consequence of modifications in material topography, whereas the current selected chemical cues inhibited cell function. Critically, in combination, topography modulated the effects of chemical environment.

Nuclear localization of HIV-1 tat functionalized gold nanoparticles.

The impermeable nature of the cell plasma membrane limits the therapeutic uses of many macromolecules and there is therefore a growing effort to circumvent this problem by designing strategies for targeted intracellular delivery. During the last decade several cell penetrating peptides, such as the HIV-1 tat peptide, have been shown to traverse the cell membrane, where integral protein transduction domains (PTDs) are responsible for their cellular uptake, and to reach the nucleus while retaining biological activity. It has since been discovered that PTDs can enable the cellular delivery of conjugated biomolecules and even nanoparticles, but nuclear delivery has remained problematic. This present study focuses on the development of water soluble, biocompatible gold nanoparticles of differing size functionalized with the HIV-1 tat PTD with the aim of producing nuclear targeting agents. The particles were subsequently tested in vitro with a human fibroblast cell line, with results demonstrating successful nanoparticle transfer across the plasma membrane, with 5 nm particles achieving nuclear entry while larger 30 nm particles are retained in the cytoplasm, suggesting entry is blocked via nuclear pores dimensions.

The interaction of human bone marrow cells with nanotopographical features in three dimensional constructs.

Until now, nanotopography has been considered in 2D construct designs. This has been due to fabrication limitations with traditional lithographic processes relying on the ability to focus radiation that will expose a radiation sensitive resist (e.g. photolithography and electron beam lithography). More recently, alternative methods that offer rapid and cheap nanofabrication have been developed; such methods include polymer demixing and colloidal lithography. Polymer demixing in 2D has relied on spin casting of polymer blends-such as polystyrene and polybromostyrene in a solvent such as toluene. As the solvent evaporates, the polymers phase separate and form nanoislands. In this study, the polymer blend solution has been blown through fine tubes and allowed to demix, thus providing 3D constructs for cell biology. The ability to fabricate in tubes may be useful in many applications, for example stents, conduits, and bone repair (when considering structures such as Haversian tubes and Volkmann's canals). As proof of concept, human osteoprogenitor cells have been used to test the cell response to the nanopatterned tubes. The results show that nanofeatures of size X, diameter Y, and spacing Z decrease cell spreading, reduce cytoskeletal organization, and increase endocytotic activity within the cells.

Age, race, diagnosis, and sodium effects on the pressor response to infused norepinephrine.

We studied the blood pressure responses to infused norepinephrine in 34 normotensive and 21 unmedicated subjects with essential hypertension. The two groups were similar in age, relative body weight, and urinary electrolyte excretion. Patients were studied on two extremes of dietary salt (200 mEq Na and 10 mEq Na per day). The dose-response curves were highly linear (p less than 0.00001) for both systolic and diastolic blood pressures. There was no evidence for an increased sensitivity to infused norepinephrine among the hypertensive subjects. On the other hand, older subjects had steeper slopes (p less than 0.005). Subjects on a high salt diet had steeper slopes than those on low salt diets (p less than 0.0025); this trend was especially apparent among blacks (p less than 0.005). Black and white hypertensive subjects responded to the high salt diet in opposite fashion: The blacks showed an increased pressor sensitivity (p less than 0.05), whereas the whites demonstrated a nonsignificant decreased pressor sensitivity. These results indicate that age, race, and salt effects must be meticulously controlled in studies of sympathetic nervous system physiology.

Genome scan for blood pressure loci in mice.

Hypertension is a complex trait of unknown cause in humans. Mice of the inbred strain BPH/2 serve as a rodent model of human hypertension and display elevated blood pressure compared with the hypotensive strain BPL/1. An F2 intercross of BPH/2 and BPL/1 and 2 backcrosses of BPL/1 with Mus spretus were used to perform interval linkage mapping for systolic blood pressure in a genome scan. Significant linkage was observed in the F2s on chromosome 10 (logarithm of the odds score [LOD]=4.9) and on chromosome 13 in the M spretus backcross (LOD=3.3), with additional suggestive LODs on chromosomes 2, 6, 8, and 18. In addition, several suggestive linkages were observed for phenotypes associated with human hypertension. Our study is the first reported genome-wide linkage scan for blood pressure genes in the mouse.

Menstrual cycle effects on catecholamine and cardiovascular responses to acute stress in black but not white normotensive women.

This study examined cardiovascular and catecholamine responses to two standardized laboratory stressors in 33 healthy age- and weight-matched black and white normotensive women (mean age, 32 years) during two phases of the menstrual cycle. Subjects were studied in a randomized order at the same time of day on two separate occasions approximately six weeks apart, once during the follicular phase (days 7 to 10 after menses) and once during the luteal phase (days 7 to 10 after the leutenizing hormone surge) of the menstrual cycle. Black women has higher systolic (P=.01) and diastolic (P=.01) pressures compared with white women. Black women showed greater diastolic pressure (P <.01) and plasma epinephrine (P <.05) responses to stress during the follicular compared with the luteal phase of the menstrual cycle; white women showed no significant changes in these variables. The findings extend the literature on race differences in responsivity to stress and indicate that in contrast to white women, reproductive hormones do influence cardiovascular and catecholamine responsivity to stress in black women.

Detecting early glaucoma by assessment of retinal nerve fiber layer thickness and visual function.

PURPOSE: To compare the abilities of scanning laser polarimetry (SLP), optical coherence tomography (OCT), short-wavelength automated perimetry (SWAP), and frequency-doubling technology (FDT) perimetry to discriminate between healthy eyes and those with early glaucoma, classified based on standard automated perimetry (SAP) and optic disc appearance. To determine the agreement among instruments for classifying eyes as glaucomatous. METHODS: One eye of each of 94 subjects was included. Healthy eyes (n = 38) had both normal-appearing optic discs and normal SAP results. Glaucoma by SAP (n = 42) required a repeatable abnormal result (glaucoma hemifield test [GHT] or corrected pattern standard deviation [CPSD] outside normal limits). Glaucoma by disc appearance (n = 51) was based on masked stereoscopic photograph evaluation. Receiver operating characteristic (ROC) curve areas, sensitivities, and specificities were calculated for each instrument separately for each diagnosis. RESULTS: The largest area under the ROC curve was found for OCT inferior quadrant thickness (0.91 for diagnosis based on SAP, 0.89 for diagnosis based on disc appearance), followed by the FDT number of total deviation plot points of < or =5% (0.88 and 0.87, respectively), SLP linear discriminant function (0.79 and 0.81, respectively), and SWAP PSD (0.78 and 0.76, respectively). For diagnosis based on SAP, the ROC curve area was significantly larger for OCT than for SLP and SWAP. For diagnosis based on disc appearance, the ROC curve area was significantly larger for OCT than for SWAP. For both diagnostic criteria, at specificities of > or =90% and > or =70%, the most sensitive OCT parameter was more sensitive than the most sensitive SWAP and SLP parameters. For diagnosis based on SAP, the most sensitive FDT parameter was more sensitive than the most sensitive SLP parameter at specificities of > or =90% and > or =70% and was more sensitive than the most sensitive SWAP parameter at specificity of > or =70%. For diagnosis based on disc appearance at specificity of > or =90%, the most sensitive FDT parameter was more sensitive than the most sensitive SWAP and SLP parameters. At specificity > or = 90%, agreement among instruments for classifying eyes as glaucomatous was poor. CONCLUSIONS: In general, areas under the ROC curve were largest (although not always significantly so) for OCT parameters, followed by FDT, SLP, and SWAP, regardless of the definition of glaucoma used. The most sensitive OCT and FDT parameters tended to be more sensitive than the most sensitive SWAP and SLP parameters at the specificities investigated, regardless of diagnostic criteria.

Internal Consistency Analysis: a method for studying the accuracy of function assessment for health outcome and quality of life evaluation.

Social, mental and physical function are major components of health outcomes and health related life quality, but the accuracy of function measurement is difficult to study rigorously. Internal Consistency Analysis (ICA) uses multiple sources of evidence from a survey interview to study the accuracy of a classification. It was developed to study function classifications for a general health outcome measure, the Quality of Well-being (QWB) scale. ICA is described and evidence of its utility in improving the classifications needed for the QWB is presented.

Effect of normal MSAFP screening on maternal age for genetic amniocentesis.

Routine maternal serum alpha-fetoprotein (MSAFP) screening for neural tube defects is considered by many to be standard obstetrical care, and recently many have encouraged this test to screen for Trisomy-21 (Down's syndrome). We questioned whether, after a normal MSAFP screen, the risk of Trisomy-21 decreases enough to warrant modifying the recommended age for genetic amniocentesis for Down's syndrome. A logistic regression was developed which, using reported values for sensitivity and specificity for MSAFP detection of Trisomy-21 and assuming a constant threshold risk in opting for amniocentesis, indicates that genetic amniocentesis for Trisomy-21 may be deferred in some women who have a normal MSAFP screening. Sensitivity analysis of varying thresholds for a normal MSAFP demonstrates that a 37 year old woman with a median MSAFP level has the same risk for Trisomy-21 as an unscreened women who is 4.5 years younger. An abnormal MSAFP is useful in screening for neural tube defects and possibly for Trisomy-21. A normal MSAFP may allow for delaying the potentially risky amniocentesis in otherwise low-risk pregnancies.

Protective effects of corticosteroids in contrast material anaphylaxis.

A prospective, randomized study of the potential protective effects of corticosteroids administered as pretreatment against contrast material (CM) reactions is reported. Patients (n = 6,763) from multiple institutions received either a single dose of steroids (32 mg of oral methylprednisolone, approximately 2 hours before CM challenge), a double dose (32 mg approximately 12 hours and 2 hours before CM challenge), or placebo. All contrast injections were with ionic media and were given intravenously. The two-dose (but not the one-dose) corticosteroid regimen provided significant protection. The effect on reaction incidences of a number of historical variables is also tabulated.

Effect of cataract and pupil size on image quality with confocal scanning laser ophthalmoscopy.

OBJECTIVE: To determine the effect of pupil size and cataract on the reproducibility and image quality obtained with confocal scanning laser ophthalmoscopy. METHODS: Three image series were obtained with a confocal scanning laser ophthalmoscope (Heidelberg Retina Tomograph, Heidelberg, Germany) before and after pupillary dilation in each of 39 subjects (8 normal subjects, 5 glaucoma suspects, and 26 patients with glaucoma). The cataract density was measured with both a lens opacity meter and the Lens Opacities Classification System III system. The image quality of each image series was subjectively scored on a scale of 0 (poor) to 9 (high) by 2 independent observers who were unaware of the cataract density and pupil size during image acquisition. The image quality and reproducibility were objectively evaluated using the SD of the mean topography image of each subject. RESULTS: The mean pupil diameter in all subjects before and after dilation was 2.5 +/- 0.8 mm and 5.8 +/- 1.4 mm, respectively. After pupillary dilation, both the mean image quality score and mean SD of the mean topography image improved (from 4.5 +/- 3.5 to 7.4 +/- 2.3 and from 48.6 +/- 18.8 microns to 35.6 +/- 15.5 microns, respectively). The quality score of the images that were obtained before pupillary dilation was associated with the pupil size and density of nuclear and posterior subcapsular cataracts. The SD of the mean topography images that were obtained before pupillary dilation increased with a decreasing pupil size (P = .003) and an increasing density of the nuclear (P < .03), cortical (P = .02), and posterior subcapsular (P = .002) opacity. CONCLUSIONS: Although pupillary dilation improved the image quality in most subjects, the improvement was sometimes small. Those subjects with small undilated pupils and/or cataracts may benefit most from pupillary dilation.

Detection of glaucoma with scanning laser polarimetry.

OBJECTIVE: To determine which retinal nerve fiber layer (RNFL) measures obtained with scanning laser polarimetry are most useful in detecting early to moderate glaucomatous visual field loss. SUBJECTS AND METHODS: One eye from 84 healthy individuals and 83 patients with early to moderate glaucomatous visual field loss (167 eyes) was assessed with a scanning laser polarimeter (Laser Diagnostic Technologies, San Diego, Calif). Three separate scans were obtained, and a baseline scan was created and used in the analyses. Integrated software (program GDx, version 1.0.02; Laser Diagnostic Technologies) was evaluated by assessing its sensitivity and specificity for detecting early and moderate glaucomatous visual field loss. Fisher linear discriminant functions also were developed in this population to assess sensitivity and specificity and were compared with the GDx analyses. RESULTS: There were statistically significant differences between the healthy and glaucomatous eyes for 14 of the 15 RNFL measures (P = .001). However, considerable overlap in measurements between groups was found. With the GDx number, the area under the receiver operator characteristic (ROC) curve was 0.78, and the sensitivity and specificity were 82% and 62%, respectively. Applying the best discriminant function using 3 variables (average thickness, ellipse modulation, and average ellipse thickness) to our study population resulted in an area under the ROC curve of 0.89 and a sensitivity and specificity of 74% and 92%, respectively. CONCLUSIONS: A combination of RNFL measures obtained using the scanning laser polarimeter improved the ability to differentiate between healthy eyes and eyes with early and moderate glaucomatous visual field loss. Analyses using GDx software did not differentiate between healthy and glaucomatous eyes as well as the discriminant analysis function did.

Discriminating between normal and glaucomatous eyes using the Heidelberg Retina Tomograph, GDx Nerve Fiber Analyzer, and Optical Coherence Tomograph.

OBJECTIVE: To compare the ability of 3 instruments, the Heidelberg Retina Tomograph (HRT), the GDx Nerve Fiber Analyzer (GDx), and the Optical Coherence Tomograph (OCT), to discriminate between healthy eyes and eyes with early to moderate glaucomatous visual field loss. SUBJECTS AND METHODS: Forty-one patients with early to moderate glaucomatous visual field loss and 50 healthy subjects were included in the study. The HRT, GDx, and OCT imaging and visual field testing were completed on 1 eye from each subject within a 6-month interval. Statistical differences in sensitivity at fixed specificities of 85%, 90%, and 95% were evaluated. In addition, areas under the receiver operating characteristic (ROC) curve were compared. RESULTS: No significant differences were found between the area under the ROC curve and the best parameter from each instrument: OCT thickness at the 5-o'clock inferior temporal position (mean +/- SE, 0.87 +/- 0.04), HRT mean height contour in the nasal inferior region (mean +/- SE, 0.86 +/- 0.04), and GDx linear discriminant function (mean +/- SE, 0.84 +/- 0.04). Twelve HRT, 2 GDx, and 9 OCT parameters had an area under the ROC curve of at least 0.81. At a fixed specificity of 90%, significant differences were found between the sensitivity of OCT thickness at the 5-o'clock inferior temporal position (71%) and parameters with sensitivities less than 52%. Qualitative assessment of stereophotographs resulted in a sensitivity of 80%. CONCLUSION: Although the area under the ROC curves was similar among the best parameters from each instrument, qualitative assessment of stereophotographs and measurements from the OCT and HRT generally had higher sensitivities than measurements from the GDx.

Race affects the decline in blood pressure with hospitalization.

Hospitalization routinely lowers blood pressure (BP). This study examined the effects of race and psychologic characteristics on this phenomenon. Data are reported from two separate cohorts of hypertensive and normotensive black and white men and women who were studied following a stay at a clinical research center where sodium intake was held constant. Blacks (N = 88), as compared to whites (N = 77), showed consistently smaller declines in systolic BP (P < .01) following hospitalization (-11.6 mm Hg SBP v -19.5 mm Hg SBP, respectively). A multiple regression model that treated BP as a function of physiologic and psychologic attributes indicated that preadmission BP level, body mass index, stress level, and anger expression were related to the drop in systolic (r2 = 65%) and diastolic (r2 = 45%) BP brought about by hospitalization (P < .0001). In blacks, high environmental stress ratings were unrelated to the change in BP with hospitalization. In contrast, whites with high environmental stress ratings lowered their BP noticeably with hospitalization. Given that the reduction in BP with hospitalization can be similar to that attained with pharmacologic therapy, these findings may have a bearing on studies examining BP in the hospital.

The effect of blood pressure cuff inflation on sleep. A polysomnographic examination.

Ambulatory blood pressure monitors are being used increasingly to define blood pressure in contexts other than the doctor's office. With increasing interest in both effects of treatment on quality of life and chronobiology of hemodynamic regulation, such monitors have also been used to define blood pressure during sleep. Unfortunately, few investigators have scrutinized the effect of such recordings on sleep, as defined precisely with polysomnography. We studied a diverse group of 12 unmedicated individuals, measuring BP by cuff inflation every 60 min. The polysomnogram was then analyzed for periods of wakefulness and arousals from sleep. Cuff inflation was associated with increased arousals (P < .0001) and wakefulness (P < .0001). In addition, subjects recalled 58% of the cuff inflations. Effects of cuff inflation on a second night of recording indicated some habituation. Nocturnal BPs measured by such techniques are still valuable, but one must have some reservations that these measures accurately reflect BP during sleep.

Which adolescent experimenters progress to established smoking in the United States.

INTRODUCTION: This study examined the adolescent smoking uptake process, specifically, the progression from experimentation to established smoking. Although adolescent smoking uptake has been described as consisting of five stages (preparation, initial trying, experimentation, regular smoking, and addiction), there is no accepted method of identifying which experimenters will proceed to become addicted. METHODS: Using a nationally representative sample of adolescents between the ages of 12 and 18 at baseline, we examined their transition from experimentation (had at least a puff but has not smoked 100 cigarettes) to established smoking (smoked at least 100 cigarettes in lifetime) four years later. RESULTS: At follow-up, 31% of the experimenters at baseline had progressed to established smoking. Baseline level of smoking experience was the strongest independent predictor of established smoking, with current experimenters (smoked in the past 30 days but less than 100 cigarettes in lifetime) having the greatest risk of progressing to established smoking compared to puffers (puffed but have not smoked a whole cigarette). Furthermore, this effect was modified by age; older current experimenters at baseline had more than double the risk of younger current experimenters of progressing to established smoking at follow-up. Absence of a firm commitment not to smoke was a significant predictor among older experimenters but not in younger experimenters. Other important predictors of the transition from experimentation to established smoking were exposure to other smokers and perceived school performance. CONCLUSIONS: We found that, even among experimenters, there is an identifiable group of adolescents who are at higher risk of progressing to established smoking that can be targeted for intervention.

Correlates of physical activity at home in Mexican-American and Anglo-American preschool children.

Twenty-two potential correlates of children's physical activity were examined. Two hundred and one Mexican-American and 146 Anglo-American families with 4-year-old children were studied. Children's physical activity was directly observed in the evening at home on 4 visits for 1 hr each time. Anglo-American children and male children were found to be more active. Demographic variables explained 11% of the variance in children's physical activity. After adjusting for demographics, 3 children's variables and 6 social-family variables did not account for significantly more variance. Five environmental variables accounted for 11% additional variance. Variables observed concurrently with physical activity, such as time spent outdoors and prompts to be active, were highly associated with children's physical activity.