Quantitative fine-tuning of photoreceptor cis-regulatory elements through affinity modulation of transcription factor binding sites.

Given the remarkable recent progress in gene therapy-based treatments for retinal disease, there is an urgent need for the development of new approaches to quantitative design and analysis of photoreceptor-specific promoters. In this study, we determined the relative binding affinity of all single-nucleotide variants of the consensus binding site of the mammalian photoreceptor transcription factor, Crx. We then showed that it is possible to use these data to accurately predict the relative binding affinity of Crx for all possible 8 bp sequences. By rationally adjusting the binding affinity of three Crx sites, we were able to fine-tune the expression of the rod-specific Rhodopsin promoter over a 225-fold range in living retinas. In addition, we showed that it is possible to fine-tune the activity of the rod-specific Gnat1 promoter over ∼275-fold range by modulating the affinity of a single Crx-binding site. We found that the action of individual binding sites depends on the precise promoter context of the site and that increasing binding affinity does not always equate with increased promoter output. Despite these caveats, this tuning approach permits quantitative engineering of photoreceptor-specific cis-regulatory elements, which can be used as drivers in gene therapy vectors for the treatment of blindness.

Vegetative buffers for fan emissions from poultry farms: 1. temperature and foliar nitrogen.

This study sought to evaluate the potential of trees planted around commercial poultry farms to trap ammonia (NH(3)), the gas of greatest environmental concern to the poultry industry. Four plant species (Norway spruce, Spike hybrid poplar, Streamco willow, and hybrid willow) were planted on eight commercial farms from 2003 to 2004. Because temperature (T) can be a stressor for trees, T was monitored in 2005 with data loggers among the trees in front of the exhaust fans (11.4 to 17.7 m) and at a control distance away from the fans (48 m) during all four seasons in Pennsylvania. Norway spruce (Picea abies) foliage samples were taken in August 2005 from one turkey and two layer farms for dry matter (DM) and nitrogen (N) analysis. The two layer farms had both Norway spruce and Spike hybrid poplar (Populus deltoides x Populus nigra) plantings sampled as well allowing comparisons of species and the effect of plant location near the fans versus a control distance away. Proximity to the fans had a clear effect on spruce foliar N with greater concentrations downwind of the fans than at control distances (3.03 vs. 1.88%; P < or = 0.0005). Plant location was again a significant factor for foliar N of both poplar and spruce on the two farms with both species showing greater N adjacent to the fans compared to the controls (3.75 vs. 2.32%; P < or = 0.0001). Pooled foliar DM of both plants was also greater among those near the fans (56.17, fan vs. 44.67%, control; P < or = 0.005). Species differences were also significant showing the potential of poplar to retain greater foliar N than spruce (3.52 vs. 2.55%; P < or = 0.001) with less DM (46.00 vs. 54.83%; P < or = 0.05) in a vegetative buffer setting. The results indicated plants were not stressed by the T near exhaust fans with mean seasonal T (13.04 vs. 13.03 degrees C, respectively) not significantly different from controls. This suggested poultry house exhaust air among the trees near the fans would not result in dormancy stressors on the plants compared to controls away from the fans.

Vegetative buffers for fan emissions from poultry farms: 2. ammonia, dust and foliar nitrogen.

This study evaluated the potential of trees planted around commercial poultry farms to trap ammonia (NH(3)) and dust or particulate matter (PM). Norway spruce, Spike hybrid poplar, hybrid willow, and Streamco purpleosier willow were planted on five commercial farms from 2003 to 2004. Plant foliage was sampled in front of the exhaust fans and at a control distance away from the fans on one turkey, two laying hen, and two broiler chicken farms between June and July 2006. Samples were analyzed for dry matter (DM), nitrogen (N), and PM content. In addition, NH(3) concentrations were measured downwind of the exhaust fans among the trees and at a control distance using NH(3) passive dosi-tubes. Foliage samples were taken and analyzed separately based on plant species. The two layer farms had both spruce and poplar plantings whereas the two broiler farms had hybrid willow and Streamco willow plantings which allowed sampling and species comparisons with the effect of plant location (control vs. fan). The results showed that NH(3) concentration h(- 1) was reduced by distance from housing fans (P < or = 0.0001), especially between 0 m (12.01 ppm), 11.4 m (2.59 ppm), 15 m (2.03 ppm), and 30 m (0.31 ppm). Foliar N of plants near the fans was greater than those sampled away from the fans for poplar (3.87 vs. 2.56%; P < or = 0.0005) and hybrid willow (3.41 vs. 3.02%; P < or = 0.05). The trends for foliar N in spruce (1.91 vs. 1.77%; P = 0.26) and Streamco willow (3.85 vs. 3.33; P = 0.07) were not significant. Pooling results of the four plant species indicated greater N concentration from foliage sampled near the fans than of that away from the fans (3.27 vs. 2.67%; P < or = 0.0001). Foliar DM concentration was not affected by plant location, and when pooled the foliar DM of the four plant species near the fans was 51.3% in comparison with 48.5% at a control distance. There was a significant effect of plant location on foliar N and DM on the two layer farms with greater N and DM adjacent to fans than at a control distance (2.95 vs. 2.15% N and 45.4 vs. 38.2% DM, respectively). There were also significant plant species effects on foliar N and DM with poplar retaining greater N (3.22 vs. 1.88%) and DM (43.7 vs. 39.9%) than spruce. The interaction of location by species (P < or = 0.005) indicated that poplar was more responsive in terms of foliar N, but less responsive for DM than spruce. The effect of location and species on foliar N and DM were not clear among the two willow species on the broiler farms. Plant location had no effect on plant foliar PM weight, but plant species significantly influenced the ability of the plant foliage to trap PM with spruce and hybrid willow showing greater potential than poplar and Streamco willow for PM(2.5)(0.0054, 0.0054, 0.0005, and 0.0016 mg cm(- 2); P < or = 0.05) and total PM (0.0309, 0.0102, 0.0038, and 0.0046 mg cm(- 2), respectively; P < or = 0.001). Spruce trapped more dust compared to the other three species (hybrid willow, poplar, and Streamco willow) for PM(10) (0.0248 vs. 0.0036 mg cm(- 2); P < or = 0.0001) and PM(> 10) (0.0033 vs. 0.0003 mg cm(- 2); P = 0.052). This study indicates that poplar, hybrid willow, and Streamco willow are appropriate species to absorb poultry house aerial NH(3)-N, whereas spruce and hybrid willow are effective traps for dust and its associated odors.

Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) with the group I intron P4-P6 domain. Thermodynamic analysis and the role of metal ions.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron's catalytic core. Previous studies suggested a model in which the protein binds first to the intron's P4-P6 domain, and then makes additional contacts with the P3-P9 domain to stabilize the two domains in the correct relative orientation to form the intron's active site. Here, we analyzed the interaction of CYT-18 with a small RNA (P4-P6 RNA) corresponding to the isolated P4-P6 domain of the N. crassa mitochondrial large subunit ribosomal RNA intron. RNA footprinting and modification-interference experiments showed that CYT-18 binds to this small RNA around the junction of the P4-P6 stacked helices on the side opposite the active-site cleft, as it does to the P4-P6 domain in the intact intron. The binding is inhibited by chemical modifications that disrupt base-pairing in P4, P6, and P6a, indicating that a partially folded structure of the P4-P6 domain is required. The temperature-dependence of binding indicates that the interaction is driven by a favorable enthalpy change, but is accompanied by an unfavorable entropy change. The latter may reflect entropically unfavorable conformational changes or decreased conformational flexibility in the complex. CYT-18 binding is inhibited at > or =125 mM KCl, indicating a strong dependence on phosphodiester-backbone interactions. On the other hand, Mg(2+) is absolutely required for CYT-18 binding, with titration experiments showing approximately 1.5 magnesium ions bound per complex. Metal ion-cleavage experiments identified a divalent cation-binding site near the boundary of P6 and J6/6a, and chemical modification showed that Mg(2+) binding induces RNA conformational changes in this region, as well as elsewhere, particularly in J4/5. Together, these findings suggest a model in which the binding of Mg(2+) near J6/6a and possibly at one additional location in the P4-P6 RNA induces formation of a specific phosphodiester-backbone geometry that is required for CYT-18 binding. The binding of CYT-18 may then establish the correct structure at the junction of the P4/P6 stacked helices for assembly of the P3-P9 domain. The interaction of CYT-18 with the P4-P6 domain appears similar to the TyrRS interaction with the D-/anticodon arm stacked helices of tRNA(Tyr).

Characterization of BCE-1, a transcriptional enhancer regulated by prolactin and extracellular matrix and modulated by the state of histone acetylation.

We have previously described a 160-bp enhancer (BCE-1) in the bovine beta-casein gene that is activated in the presence of prolactin and extracellular matrix (ECM). Here we report the characterization of the enhancer by deletion and site-directed mutagenesis, electrophoretic mobility shift analysis, and in vivo footprinting. Two essential regions were identified by analysis of mutant constructions: one binds C/EBP-beta and the other binds MGF/STAT5 and an as-yet-unidentified binding protein. However, no qualitative or quantitative differences in the binding of these proteins were observed in electrophoretic mobility shift analysis using nuclear extracts derived from cells cultured in the presence or absence of ECM with or without prolactin, indicating that prolactin- and ECM-induced transcription was not dependent on the availability of these factors in the functional cell lines employed. An in vivo footprinting analysis of the factors bound to nuclear chromatin in the presence or absence of ECM and/or prolactin found no differences in the binding of C/EBP-beta but did not provide definitive results for the other factors. Neither ECM nor prolactin activated BCE-1 in transient transfections, suggesting that the chromosomal structure of the integrated template may be required for ECM-induced transcription. Further evidence is that treatment of cells with inhibitors of histone deacetylase was sufficient to induce transcription of integrated BCE-1 in the absence of ECM. Together, these results suggest that the ECM induces a complex interaction between the enhancer-bound transcription factors, the basal transcriptional machinery, and a chromosomally integrated template responsive to the acetylation state of the histones.

A tyrosyl-tRNA synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain). To investigate how CYT-18 stabilizes the active RNA structure, we used an Escherichia coli genetic assay based on the phage T4 td intron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long-range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain. Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7. CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site. Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees. The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in the td intron. Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron's active site.

Patterns of communication about AIDS among Hispanic and Anglo adolescents.

This study examined exposure to AIDS information from mass media and interpersonal communication among a nonprobability sample of Hispanic and Anglo lower middle-class adolescents. Subjects were drawn predominantly from community health clinics, word of mouth recruiting, public service announcements, churches, schools, and health fairs in San Diego County. Both Hispanic (n = 220) and Anglo (n = 159) youths reported substantial exposure to information about AIDS from both mass media and interpersonal communication. Hispanics watched more general television than Anglos, but had less exposure to newspapers and interpersonal communication with friends. Communication among friends was most consistently related to sociodemographic variables, with older, higher status persons and girls communicating to the greatest extent. Boys reported the greatest communication with family. Anglo youths had greater exposure to information about AIDS and condoms regardless of the medium of communication, while Hispanic adolescents had greater exposure to information on risks of IV drug use. Of the three main types of mass media, print and radio provided the most exposure to AIDS information. It is important for preventive medicine practitioners to exploit these differences in communication patterns when planning preventive intervention strategies that target specific adolescent populations.

Tenascin-C inhibits extracellular matrix-dependent gene expression in mammary epithelial cells. Localization of active regions using recombinant tenascin fragments.

The physiological role of tenascin in vivo has remained obscure. Although tenascin is regulated in a stage and tissue-dependent manner, knock-out mice appear normal. When tenascin expression was examined in the normal adult mouse mammary gland, little or none was present during lactation, when epithelial cells actively synthesize and secrete milk proteins in an extracellular matrix/lactogenic hormone-dependent manner. In contrast, tenascin was prominently expressed during involution, a stage characterized by the degradation of the extracellular matrix and the subsequent loss of milk production. Studies with mammary cell lines indicated that tenascin expression was high on plastic, but was suppressed in the presence of the laminin-rich, Engelbreth-Holm-Swarm (EHS) tumour biomatrix. When exogenous tenascin was added together with EHS to mammary epithelial cells, beta-casein protein synthesis and steady-state mRNA levels were inhibited in a concentration-dependent manner. Moreover, this inhibition by tenascin could be segregated from its effects on cell morphology. Using two beta-casein promoter constructs attached to the chloramphenicol acetyltransferase reporter gene we showed that tenascin selectively suppressed extracellular matrix/prolactin-dependent transcription of the beta-casein gene in three-dimensional cultures. Finally, we mapped the active regions within the fibronectin type III repeat region of the tenascin molecule that are capable of inhibiting beta-casein protein synthesis. Our data are consistent with a model where both the loss of a laminin-rich basement membrane by extracellular matrix-degrading enzymes and the induction of tenascin contribute to the loss of tissue-specific gene expression and thus the involuting process.

A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of beta-casein gene expression.

Lactogenic hormones and extracellular matrix (ECM) act synergistically to regulate beta-casein expression in culture. We have developed a functional subpopulation of the mouse mammary epithelial cell strain COMMA-1D (designated CID 9), which expresses high level of beta-casein, forms alveolar-like structures when plated onto the EHS tumor-derived matrix, and secretes beta-casein unidirectionally into a lumen. We have further shown that ECM- and prolactin-dependent regulations of beta-casein occur mainly at the transcriptional level and that 5' sequences play an important role in these regulations. To address the question of the nature of the DNA sequence requirements for such regulation, we analyzed the bovine beta-casein gene promoter in these cells. We now have located a 160-bp transcriptional enhancer (BCE1) within the 5' flanking region of the beta-casein gene. Using functional assays, we show that BCE1 contains responsive elements for prolactin- and ECM-dependent regulation. BCE1 placed upstream of a truncated and inactive beta-casein promoter (the shortest extending from -89 to +42 bp with regard to the transcription start site) reconstitutes a promoter even more potent than the intact promoter, which contains BCE1 in its normal context more than 1.5 kb upstream. This small fusion promoter also reconstitutes the normal pattern of regulation, including a requirement for both prolactin and ECM and a synergistic action of prolactin and hydrocortisone. By replacing the milk promoter with a heterologous viral promoter, we show that BCE1 participates in the prolactin- and ECM-mediated regulation.

Gene transfer in primary cultures of human hepatocytes.

Using liposomes as the mediator of DNA transfer, we were successful in the transfection of human hepatocytes isolated from surgical samples with an E. coli beta-galactosidase gene (beta-gal). A comparison of transfection efficiency showed that of the four promoters used, cytomegalovirus (CMV) promoter yielded higher transfection efficiencies than Rous sarcoma virus (RSV), Simian virus-40 (SV-40) and human alpha-1 antitrypsin (AAT) promoters. These studies represent the first report on the successful transfection of primary cultures of human hepatocytes.

Paradoxical effects of blood alcohol concentration charts.

BACKGROUND: This study was designed to determine the association between alcohol drinking and the possession of blood alcohol concentration charts. METHOD: A total of 30 college-age subjects participated in the study. Subjects were randomly assigned to either an experimental or a control group. All subjects answered a short entrance questionnaire to determine their height and weight. Those assigned to the experimental group were supplied a copy of a blood alcohol concentration chart and instructed in its use. The volume of alcoholic beverages consumed was surreptitiously counted for all subjects. Prior to leaving the premises the subjects completed an exit questionnaire which asked them to estimate the amount of alcohol they had consumed, whether they had driven to the pub, and whether they intended to drive away. The time spent in the pub was noted for each subject. RESULTS: Among those in the control group there was a tendency to overestimate the volume consumed, and for those in the experimental group, a tendency to more accurately estimate their consumption. Those given blood alcohol concentration charts consumed alcohol at a significantly higher rate than did those in the control group. CONCLUSION: A likely explanation for this outcome is that the chart served as a stimulus to prompt a drinker to more quickly achieve a blood alcohol level consistent with his/her drinking expectancies.

Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5' sequences in stably transfected mouse mammary cells.

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-1D has been developed in which more than 35% of the cells express beta-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete beta-casein unidirectionally into a lumen. These cells were stably transfected with a series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine beta-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency (greater than 150-fold induction in some cases). This regulation occurred primarily at the transcriptional level and was dependent on the length of the 5' flanking region of the beta-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous beta-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.

Determination of peak trabecular bone density: interplay of dietary fiber, carbohydrate, and androgens.

To elucidate mechanisms linking nutrition and sex hormones to magnitude of peak trabecular bone density, we studied 11 normal women aged 19-21 y. Trabecular bone density was related inversely to dietary fiber (r = -0.69, p = 0.02) and carbohydrate (r = -0.70, p = 0.02) and directly to serum free-and-albumin-bound testosterone (fab T) (r = -0.70, p = 0.02) and total testosterone (total T) (r = 0.66, p = 0.03). Dietary fiber was correlated negatively with fab T (r = -0.74, p = 0.009), total T (r = -0.70, p = 0.02), and androstenedione (Adione) (r = -0.72, p = 0.01). Controlling for the effect of fab T or Adione weakened the relationship between dietary fiber and bone density and the relationship was no longer statistically significant. Conversely, controlling for sex hormones did not abolish the effect of carbohydrate on bone density. The contributions of fab T and carbohydrate to bone density were independent. These results suggest that dietary fiber may depress serum androgens which in turn decrease trabecular bone density. Carbohydrate may also depress bone density but independently of sex steroid hormones.

Case report: unresponsive choroidal metastases.

1. The most common tumor of the choroid is metastatic tumor, with the lung as the primary site. Diagnosis may be difficult in distinguishing a choroidal melanoma from a metastatic lesion. 2. Careful metastatic evaluation may not identify an original primary tumor. Tests may not be sensitive enough to disclose adenocarcinoma until it has reached an advanced stage. 3. External beam radiation may not always be successful in treating choroidal metastases.

In vitro evaluation of the cytotoxic potential of a novel man-made fiber, calcium sodium metaphosphate fiber (Phosphate Fiber).

As part of a comprehensive effort to evaluate the toxicological potential of calcium sodium metaphosphate fiber (Phosphate Fiber), the in vitro cytotoxicity of the fiber in cultured cells was studied. Two pulmonary-derived cell systems (rat alveolar macrophages, RAM; rat lung epithelial cells, LEC) and an established cell line (Chinese hamster ovary, CHO) were used. Release of lactate dehydrogenase (LDH) was used as an endpoint for cytotoxicity for all three cell types. In addition, inhibition of colony formation was used for CHO cells. The cytotoxicity of Phosphate Fiber was compared to a variety of mineral dusts and fibers including chrysotile asbestos, crocidolite asbestos, two glass fibers, calcium sulfate fiber, titanium dioxide, as well as the nonfibrous raw material, calcium sodium metaphosphate glass. Results with all three cell culture systems demonstrated that the Phosphate Fiber was less cytotoxic than the two asbestos fibers, similar in cytotoxicity to the glass fibers, and more cytotoxic than the calcium sulfate fiber and titanium dioxide. To further investigate the cytotoxicity of the Phosphate Fiber, it was fractionated by sedimentation into small and large fibers. The small Phosphate Fiber was found to be more cytotoxic and the large Phosphate Fiber to be less cytotoxic than the unfractionated Phosphate Fiber. The in vitro data suggest that Phosphate Fiber is less cytotoxic than asbestos, but further determination of safety can only be made after the in vivo data have been obtained.

Effect of declining renal function on bone density in aging women.

The factors that are responsible for trabecular bone loss in aging women are not completely understood. To evaluate declining renal function as a possible factor, we studied 19 Caucasian women (average age 67) who were from 6 to 41 years postmenopausal. Trabecular bone density was quantitated by computerized tomography of the spine. Serum calcium, phosphorus, and creatinine were normal in all subjects. Creatinine clearance averaged 74 ml/min (range 38-122), decreased with age (r = -0.60, P = 0.003), and was inversely related to serum creatinine (r = -0.51, P = 0.01). Bivariate regression demonstrated that bone density decreased with age (r = -0.59, P = 0.004); controlling for the effect of creatinine clearance weakened this correlation to r = -0.45 (P = 0.03); controlling additionally for 1,25-dihydroxyvitamin D [1,25(OH)2D] and parathyroid hormone (PTH) reduced the correlation coefficient to r = -0.34 (P = 0.11). Bone density also decreased in direct proportion to the decrement in creatinine clearance (r = 0.44, P = 0.03); controlling for the effects of 1,25(OH)2D and PTH reduced this correlation coefficient to r = 0.34 (P = 0.11). These results suggest that occult renal insufficiency may contribute to bone loss in aging women, and that this effect may be mediated in part by 1,25(OH)2D and PTH. In this age group renal function should be assessed by measuring creatinine clearance rather than the serum creatinine concentration since renal insufficiency can be masked by apparently normal circulating creatinine levels.

A comparison of the risk of vertebral fracture in menopausal osteopenia and other metabolic disturbances.

The risk of atraumatic compression fracture in postmenopausal women increases as vertebral trabecular bone density decreases. To determine whether the risk is similar for patients who have other metabolic disorders, we compared eight-nine patients who had various disturbances affecting bone and sixty-three postmenopausal women who had no evidence of underlying disease. Trabecular bone density was measured by quantitative computed tomography of the lumbar spine. The relationship between frequency of fracture and bone density was modeled mathematically with spline threshold, quadratic polynomial, and decaying exponential functions. Analysis of covariance showed that the diagnostic category did not influence the relationship between frequency of fracture and bone density in any of the three models. We concluded that the risk of atraumatic compression fracture, as assessed by measurement of vertebral trabecular bone density using quantitative computerized tomography, is independent of the underlying metabolic disturbance.