The effects of pastoral intensification on the feeding interactions of generalist predators in streams.

Land-use change can alter trophic interactions with wide-ranging functional consequences, yet the consequences for aquatic food webs have been little studied. In part, this may reflect the challenges of resolving the diets of aquatic organisms using classical gut contents analysis, especially for soft-bodied prey. We used next-generation sequencing to resolve prey use in nearly 400 individuals of two predatory invertebrates (the Caddisfly, Rhyacophila dorsalis, and the Stonefly Dinocras cephalotes) in streams draining land with increasingly intensive livestock farming. Rhyacophila dorsalis occurred in all streams, whereas D. cephalotes was restricted to low intensities, allowing us to test whether: (i) apparent sensitivity to agriculture in the latter species reflects a more specialized diet and (ii) diet in R. dorsalis varied between sites with and without D. cephalotes. DNA was extracted from dissected gut contents, amplified without blocking probes and sequenced using Ion Torrent technology. Both predators were generalists, consuming 30 prey taxa with a preference for taxa that were abundant in all streams or that increased with intensification. Where both predators were present, their diets were nearly identical, and R. dorsalis's diet was virtually unchanged in the absence of D. cephalotes. The loss of D. cephalotes from more intensive sites was probably due to physicochemical stressors, such as sedimentation, rather than to dietary specialization, although wider biotic factors (e.g., competition with other predatory taxa) could not be excluded. This study provides a uniquely detailed description of predator diets along a land-use intensity gradient, offering new insights into how anthropogenic stressors affect stream communities.

Experimental characterization of vertical-axis wind turbine noise.

Vertical-axis wind turbines are wind-energy generators suitable for use in urban environments. Their associated noise thus needs to be characterized and understood. As a first step, this work investigates the relative importance of harmonic and broadband contributions via model-scale wind-tunnel experiments. Cross-spectra from a pair of flush-mounted wall microphones exhibit both components, but further analysis shows that the broadband dominates at frequencies corresponding to the audible range in full-scale operation. This observation has detrimental implications for noise-prediction reliability and hence also for acoustic design optimization.

ACVRL1 germinal mosaic with two mutant alleles in hereditary hemorrhagic telangiectasia associated with pulmonary arterial hypertension.

Germline mutations in genes encoding members of the transforming growth factor-ß (TGF-ß)/bone morphogenetic protein (BMP) superfamily are causal for two hereditary vascular disorders, hereditary hemorrhagic telangiectasia (HHT) and heritable pulmonary arterial hypertension (PAH). When the two diseases coexist, activin A receptor type II-like kinase-1 (ACVRL1) gene mutations are usually identified. We report a remarkable ACVRL1 germinal and somatic mosaicism characterized by the presence of two distinct mutant alleles and a non-mutant ACVRL1 allele in a woman diagnosed with PAH at the age 40. She also met the Curaçao diagnostic criteria for HHT based on additional findings of telangiectases, epistaxis and arteriovenous malformations. Mutation analysis of ACVRL1 identified two adjacent heterozygous deleterious mutations within exon 10: c.1388del (p.Gly463fsX2) and c.1390del (p.Leu464X) in a region enriched by mutation-associated DNA motifs. The mother transmitted the c.1388del to one child and the c.1390del to two children confirming germinal mosaicism. Allele-specific polymerase chain reaction analysis showed that c.1388del is the predominant mutation in lymphocytes of the index case. Haplotype analysis revealed that both mutant alleles have a common chromosomal origin which is distinct from that of the mother's non-mutant ACVRL1 allele. These distinct mutant alleles in tissues and germline could have arisen by DNA structure-mediated events occurring in the early stages of the mother's embryogenesis, prior to the segregation of her germline, which ultimately led to the independent transmission of each allele. These highlight the complexity of genomic events occurring during early embryogenesis and the consequences of mutational mosaicism upon pathogenic variability.

Trinucleotide repeat DNA structures: dynamic mutations from dynamic DNA.

Models for the disease-associated expansion of (CTG)n.(CAG)n, (CGG)n.(CCG)n, and (GAA)n.(TTC)n trinucleotide repeats involve alternative DNA structures formed during DNA replication, repair and recombination. These repeat sequences are inherently flexible and can form a variety of hairpins, intramolecular triplexes, quadruplexes, and slipped-strand structures that may be important intermediates and result in their genetic instability.

Estrogen receptor and hormone responsiveness of medullary thyroid carcinoma cells in continuous culture.

We have examined the estrogen responsiveness and estrogen receptor in medullary thyroid carcinoma using a model of an established human cell line, TT. TT cells bind [3H]estradiol with high affinity. Scatchard analysis reveals a single class of binding site with a concentration of 173 fmol/10(6) cells and a dissociation constant of 2.1 x 10(-9) M, values which are comparable to those of a well established model cell line for estrogen responsiveness, MCF-7 human breast cancer cell line. Estradiol in physiological concentrations moderately stimulated TT cell proliferation, whereas in pharmacological concentrations it markedly inhibited cell growth. [3H]Thymidine incorporation into acid-insoluble material was also stimulated following a 5-day treatment with 5 x 10(-9) M estradiol. Tamoxifen at a concentration of 1 microM reduced cell proliferation by 43-48% after 5-7 days of treatment. The growth suppression induced by tamoxifen was reversed by addition of 10 nM estradiol. This is the first report of estrogen growth stimulation and tamoxifen growth inhibition of a tumor cell line derived from human medullary thyroid carcinoma.

Plasmids bearing mammalian DNA-replication origin-enriched (ors) fragments initiate semiconservative replication in a cell-free system.

Four plasmids containing monkey (CV-1) origin-enriched sequences (ors), which we have previously shown to replicate autonomously in CV-1, COS-7 and HeLa cells (Frappier and Zannis-Hadjopoulos (1987) Proc. Natl. Acad. Sci. USA 84, 6668-6672), were found to replicate in an in vitro replication system using HeLa cell extracts. De novo site-specific initiation of replication on plasmids required the presence of an ors sequence, soluble low-salt cytosolic extract, poly(ethylene glycol), a solution containing the four standard deoxyribonucleoside triphosphates and an ATP regenerating system. The major reaction products migrated as relaxed circular and linear plasmid DNAs, both in the presence and absence of high-salt nuclear extracts. Inclusion of high-salt nuclear extract was required to obtain closed circular supercoiled molecules. Replicative intermediates migrating slower than form II and topoisomers migrating between forms II and I were also included among the replication products. Replication of the ors plasmids was not inhibited by ddTTP, an inhibitor of DNA polymerase beta and gamma, and was sensitive to aphidicolin indicating that DNA polymerase alpha and/or delta was responsible for DNA synthesis. Origin mapping experiments showed that early in the in vitro replication reaction, incorporation of nucleotides occurs preferentially at ors-containing fragments, indicating ors specific initiation of replication. In contrast, the limited incorporation of nucleotides into pBR322, was not site specific. The observed synthesis was semiconservative and appeared to be bidirectional.

Can characteristics of a health care system mitigate ethnic bias in access to cardiovascular procedures? Experience from the Military Health Services System.

OBJECTIVES: This study sought to investigate the independent effect of ethnicity on the utilization of invasive cardiac procedures after acute myocardial infarction (AMI). BACKGROUND: The precise role of ethnicity in access to cardiovascular procedures is unknown, particularly because of difficulty in isolating ethnicity from financial and other socioeconomic factors. We conducted a retrospective analysis of the use of cardiac catheterization and coronary revascularization procedures after AMI in military health care beneficiaries. The Military Health Services System (MHSS) ensures equal access to care in an environment without financial incentives for procedural utilization; furthermore, socioeconomic differences between patients beyond ethnicity are minimized. METHODS: Data were analyzed from the Civilian External Peer Review Program representing abstracted chart reviews from 125 military health care facilities worldwide for all patients (1,208 white; 233 nonwhite [155 black]) with the principal or secondary diagnosis of AMI from March to September 1993. RESULTS: Rates of cardiac catheterization were similar in white and nonwhite patients (34.8 vs. 39.1%, p = 0.21). After controlling for age, gender, cardiovascular risk factors and AMI variables, including infarct size and other risk markers, there were no differences in the use of this procedure during the AMI admission in comparisons of white versus nonwhite patients (estimated odds ratio [OR] 0.96, 95% confidence interval [CI] 0.69 to 1.34) and white versus black patients (OR 1.19, 95% CI 0.80 to 1.78). However, white patients were significantly more likely than nonwhite patients to be "considered" for future cardiac catheterization (OR 1.77, 95% CI 1.19 to 2.61). Coronary revascularization within 180 days was not significantly affected by race in white versus nonwhite (OR 0.90, 95% CI 0.59 to 1.39) and white versus black patients (OR 1.11, 95% CI 0.65 to 1.89). Outcomes (30- and 180-day mortality and readmission rates) were similar for all race groups. CONCLUSIONS: There is a limited relation between ethnicity and the use of invasive cardiac procedures in the MHSS. These data raise the promise that characteristics of a health care system can mitigate ethnic bias in medicine.

Unexpected formation of parallel duplex in GAA and TTC trinucleotide repeats of Friedreich's ataxia.

The onset and progress of Friedreich's ataxia (FRDA) is associated with the genetic instability of the (GAA).(TTC) trinucleotide repeats located within the frataxin gene. The instability of these repeats may involve the formation of an alternative DNA structure. Poly-purine (R)/poly-pyrimidine (Y) sequences typically form triplex DNA structures which may contribute to genetic instability. Conventional wisdom suggested that triplex structures formed by these poly-purine (R)/poly-pyrimidine (Y) sequences may contribute to their genetic instability. Here, we report the characterization of the single-stranded GAA and TTC sequences and their mixtures using NMR, UV-melting, and gel electrophoresis, as well as chemical and enzymatic probing methods. We show that the FRDA GAA/TTC, repeats are capable of forming various alternative structures. The most intriguing is the observation of a parallel (GAA).(TTC) duplex in equilibrium with the antiparallel Watson-Crick (GAA).(TTC) duplex. We also show that the GAA strands form self-assembled structures, whereas the TTC strands are essentially unstructured. Finally, we demonstrate that the FRDA repeats form only the YRY triplex (but not the RRY triplex) at neutral pH and the complete formation of the YRY triplex requires the ratio of GAA to TTC strand larger than 1:2. The structural features presented here and in other studies distinguish the FRDA (GAA)¿(TTC) repeats from the fragile X (CGG).CCG), myotonic dystrophy (CTG).(CAG) and the Huntington (CAG).(CTG) repeats.

The contribution of cis-elements to disease-associated repeat instability: clinical and experimental evidence.

Alterations in the length (instability) of gene-specific microsatellites and minisatellites are associated with at least 35 human diseases. This review will discuss the various cis-elements that contribute to repeat instability, primarily through examination of the most abundant disease-associated repetitive element, trinucleotide repeats. For the purpose of this review, we define cis-elements to include the sequence of the repeat units, the length and purity of the repeat tracts, the sequences flanking the repeat, as well as the surrounding epigenetic environment, including DNA methylation and chromatin structure. Gender-, tissue-, developmental- and locus-specific cis-elements in conjunction with trans-factors may facilitate instability through the processes of DNA replication, repair and/or recombination. Here we review the available human data that supports the involvement of cis-elements in repeat instability with limited reference to model systems. In diverse tissues at different developmental times and at specific loci, repetitive elements display variable levels of instability, suggesting vastly different mechanisms may be responsible for repeat instability amongst the disease loci and between various tissues.

Giant lymph node hyperplasia involving the thymus with associated nephrotic syndrome and myelofibrosis.

Giant lymph node hyperplasia (GLH) is an unusual form of benign lymphoid hyperplasia which, although it often occurs in the mediastinum, has not previously been reported to involve the thymus. In this report, a case of GLH with involvement of the thymus and with associated nephrotic syndrome and myelofibrosis is presented. The significance of the association of these conditions with GLH is discussed, including the possibility that some or all of the patient's abnormalities may have been related via an abnormal immune mechanism.

Dual-balloon angioplasty of recoarctation of the aorta.

Percutaneous balloon angioplasty is a successful method for treating recoarctation of the aorta after surgical repair. The procedure is usually performed with small balloons on 4 or 5 French (Fr) shafts (Gruentzig or Cook) in infants or large balloons on 8 or 9 Fr shafts (Meditech or Mansfield) in older children. In certain children, however, the 8 or 9 Fr shafts may be too large for insertion in the femoral artery, and the 4 or 5 Fr shafts may not carry a balloon large enough to effectively dilate the area of restenosis. We describe a case involving a 9-kg infant in whom recoarctation of the aorta was successfully treated with two small side-by-side balloon angioplasty systems.

Establishing a self-perpetuating American Heart Association/Advanced Cardiac Life Support program in West Germany.

Sudden cardiac death is one of the major health problems in industrialized nations. The American Heart Association-sponsored Basic Life Support and Advanced Cardiac Life Support (ACLS) programs provide consensus training programs geared to teach local persons and health care providers the techniques of CPR and advanced life support, respectively. In April 1988, the staff of the U.S. Army Hospital Berlin conducted an ACLS provider course for German physicians. Fourteen students attended the first course; nine passed, and two did not complete the course. Two more courses have been completed. The enthusiasm expressed by the German physicians led to the nomination of one of the physicians for an associate faculty position, and plans for an instructor course of German physicians are under way.

DNA methylation and replication: implications for the "deletion hotspot" region of FMR1.

Expansion and hyper-methylation of a CGG repeat tract are the main causes of fragile X syndrome (FRAXA). In some rare instances, FRAXA patients harbor not only an expanded CGG tract, but a deletion encompassing the CGG repeat and flanking sequences as well. Through the use of an SV40 primate replication system, it was possible to determine that CpG methylation and DNA replication may actually mediate the formation of these rare events. Also, the genetically stabilizing AGG interruptions can be lost by replication-mediated CGG deletions.

Health promotion and health education: the emerging role of the private sector.

The climate is right for new ventures in health education, especially those in which corporate leaders join forces with other members of the communities where they do business. Working with the public sector and other members of the private sector--with schools, universities, health departments, churches, professional associations, voluntary agencies, and foundations--corporations can become a vital new force in supporting health education not only of their own employees but also of children and adults and the public at large.

Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci.

Most models proposed to explain the disease-associated expansion of (CTG)n.(CAG)n and (CGG)n.(CCG)n trinucleotide repeats include the formation of slipped strand DNA structures during replication; however, physical evidence for these alternative DNA secondary structures has not been reported. Using cloned fragments from the myotonic dystrophy (DM) and fragile X syndrome (FRAXA) loci containing normal, premutation, and full mutation lengths of repeats, we report the formation of novel alternative DNA secondary structures that map within the repeat tracts during reannealing of complementary strands, containing equal lengths of repeats, into linear duplex DNA molecules. Linear duplex DNA molecules containing these alternative DNA secondary structures are characterized by reduced electrophoretic mobilities in polyacrylamide gels. These alternative secondary structures are stable at physiological ionic strengths and to temperatures up to at least 55 degrees C. Following reduplexing, the CAG strand of the (CTG)n.(CAG)n repeats is preferentially sensitive to mung bean nuclease, suggesting the presence of single-stranded DNA in the alternative DNA structure. For (CTG)17, which is a repeat length found in normal individuals, less than 3% of the DNA molecules formed alternative DNA structures upon reduplexing. DNA molecules containing (CTG)50 or (CTG)255, which represent premutation and full mutation lengths of triplet repeats, respectively, formed a heterogeneous population of alternative DNA structures in >50% of DNA molecules. The complexity of the structures formed increased with the length of the triplet repeat. The relationship between repeat length and the propensity of formation and complexity of the novel structures correlates with the effect of repeat length on genetic instability in human diseases. These are the first results consistent with the existence of slipped strand DNA structures. The potential involvement of these structures, which we term S-DNA, in the gentic instability of triplet repeats is discussed.

Stability of triplet repeats of myotonic dystrophy and fragile X loci in human mutator mismatch repair cell lines.

At least nine human genetic diseases, including myotonic dystrophy (DM) and fragile X syndrome have been associated with the expansion of CTG or CGG trinucleotide repeats within the disease loci. Little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. Mutations in human mismatch repair genes are associated with the increased polymorphism of many microsatellites, including dinucleotide repeats. The effect of mutations in two mismatch repair genes on the size of trinucleotide repeats in the DM and FRAXA loci has been analyzed. PCR and Southern analysis of the triplet repeat regions of the DM and fragile X mental retardation (FRAXA) loci in cell lines HTC116 and LoVo, which contain mutations in both alleles of the hMLH1 and hMSH2 genes, respectively, indicated that the size of the endogenous (CTG)n and (CGG)n tracts fall within the range observed in the normal population. This suggests that mutations in hMLH1 or hMSH2 do not result in the instability of CTG or CGG tracts to the levels observed in individuals with myotonic dystrophy or fragile X syndrome.

A novel type of interaction between cruciform DNA and a cruciform binding protein from HeLa cells.

We recently identified and enriched a protein (CBP) from HeLa cells with binding specificity for cruciform-containing DNA. We have now studied the interaction of CBP with stable cruciform DNA molecules containing the 27 bp palindrome of SV40 on one strand and an unrelated 26 bp palindrome on the other strand by hydroxyl radical footprinting. The CBP-DNA interaction is localized to the four-way junction at the base of the cruciforms. CBP appears to interact with the elbows of the junctions in an asymmetric fashion. Upon CBP binding, structural distortions were observed in the cruciform stems and in a DNA region adjacent to the junction. These features distinguish CBP from other cruciform binding proteins, which bind symmetrically and display exclusively either contacts with the DNA backbone or structural alterations in the DNA.

Deletion analysis of minimal sequence requirements for autonomous replication of ors8, a monkey early-replicating DNA sequence.

We have generated a panel of deletion mutants of ors8 (483 bp), a mammalian autonomously replicating DNA sequence, previously isolated by extrusion of nascent monkey (CV-1) DNA from replication bubbles active at the onset of S phase. The deletion mutants were tested for replication function by the DpnI resistance assay, in vivo, after transfection into HeLa cells, and in vitro. An internal fragment of 186-bp that is required for autonomous replication function of ors8 was identified. This fragment, when subcloned into pBR322 and similarly tested, was capable of autonomous replication in vivo and in vitro. The 186-bp fragment contains several repeated sequence motifs, such as the ATTA and ATTTAT motifs, occurring three and five times, respectively, the sequences TAGG and TAGA, occurring three and seven times, respectively, two 5'-ATT-3' repeats, a 44-bp imperfect inverted repeat (IR) sequence, and an imperfect consensus binding element for the transcription factor Oct-1. A measurable sequence-directed DNA curvature was also detected, coinciding with the AT-rich regions of the 186-bp fragment.

Pressure changes in the eye due to an injection of inert gases: a theoretical model.

In the repair of retinal tears and detachments, the vitreous humor is often completely replaced with a temporary mixture of gases, one of which is not normally found in the bloodstream. The resulting bubble can then support the healing retina. There is diffusion of gases, however, from the bubble into the bloodstream and vice versa. This alters the intraocular pressure, with possible adverse consequences, as the intraocular pressure must be maintained within a certain range for the procedure to be successful. A simple model has been developed to predict the evolution of intraocular pressure over time, given a certain initial mixture of injected gases. This model could be useful in determining what mixture to use to support effectively and safely a healing retina.