Analyzing Chromatin Structure and Transcription Factor Binding in Yeast

The study of chromatin, once thought to be a purely structural matrix serving to compact the DNA of the genome into the nucleus, is of increasing value for our understanding of how DNA functions in the cell. This article provides two basic procedures for the study of chromatinin vivo.The first is a DNase I-based method for the treatment of isolated nuclei to resolve the chromatin structure of a particular region; the second employs dimethyl sulfate footprinting of whole cellsin vivoto determine the binding of factors tociselements in the locus of interest. Specific examples illustrating the techniques described are given from our work on the regulation of the yeastPHO8gene, but have also been successfully and reliably applied to the study of many other yeast loci. These procedures make it possible to correlate the binding of atransactivator with an altered or perturbed chromatin organization at a specific locus.     

Biodegradation of cyanides, cyanates and thiocyanates to ammonia and carbon dioxide by immobilized cells of Pseudomonas putida

Pseudomonas putida utilizes cyanide as the sole source of carbon and nitrogen. Agar, alginate, and carrageenan were screened as the encapsulating matrices for P. putida. Alginate-immobilized cells of P. putida degraded sodium cyanide (NaCN) more efficiently than non-immobilized cells or cells immobilized in agar or carrageenan. The end products of biodegradation of cyanide were identified as ammonia (NH₃) and carbon dioxide (CO₂). These products changed the medium pH. In bioreactors, the rate of cyanide degradation increased with an increase in the rate of aeration. Maximum utilization of cyanide was observed at 200 ml min⁻¹ of aeration. Immobilized cells of P. putida degraded cyanides, cyanates and thiocyanates to NH₃ and CO₂. Use of Na[¹⁴C]-CN showed that 70% of carbon of Na[¹⁴C]-CN was converted into ¹⁴CO₂ and only 10% was associated with the cell biomass. The substrate-dependent kinetics indicated that the K _m and V _max values of P. putida for the substrate, NaCN were 14 mM and 29 nmol of oxygen consumed mg protein⁻¹ min⁻¹ respectively. Received 29 January 1996/ Accepted in revised form 19 September 1997     

Prevalence of Epstein-Barr virus (EBV) antibodies in primate stocks of zoological gardens

Abstract: Examination of 387 serum samples from 41 primate species with two different ELISAs for the presence of IgG-antibodies against Epstein-Barr virus. Antibodies were detected in 15 out of 32 species of Old World primates and none in six species of New World primates by screening ELISA (Enzygnost, Behringwerke AG, Marburg), a testkit for human diagnostics. To avoid species-dependent factors which could influence the sensitivity of the Enzygnost assay, a competition ELISA was established. The modified test assessed antibodies in all species of Old World primates and three species of the New World primates.     

Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT‐SWATH mass spectrometry

The amount of sample available for clinical and biological proteomic research is often limited and thus significantly restricts clinical and translational research. Recently, we have integrated pressure cycling technology (PCT) assisted sample preparation and SWATH‐MS to perform reproducible proteomic quantification of biopsy‐level tissue samples. Here, we further evaluated the minimal sample requirement of the PCT‐SWATH method using various types of samples, including cultured cells (HeLa, K562, and U251, 500 000 to 50 000 cells) and tissue samples (mouse liver, heart, brain, and human kidney, 3–0.2 mg). The data show that as few as 50 000 human cells and 0.2–0.5 mg of wet mouse and human tissues produced peptide samples sufficient for multiple SWATH‐MS analyses at optimal sample load applied to the system. Generally, the reproducibility of the method increased with decreasing tissue sample amounts. The SWATH maps acquired from peptides derived from samples of varying sizes were essentially identical based on the number, type, and quantity of identified peptides. In conclusion, we determined the minimal sample required for optimal PCT‐SWATH analyses, and found smaller sample size achieved higher quantitative accuracy.     

Left Ventricular Systolic Dysfunction in Asymptomatic Marfan Syndrome Patients Is Related to the Severity of Gene Mutation: Insights from the Novel Three Dimensional Speckle Tracking Echocardiography

Background

In asymptomatic Marfan syndrome (MFS) patients we evaluated the relationship between the types of fibrillin-1 (FBN1) gene mutation and possible altered left ventricular (LV) function as assessed by three-dimensional speckle tracking echocardiography (3D-STE).

Methods and Results

Forty-five MFS patients (mean age 24±15 years) and 40 age-matched healthy controls were studied. Genetic evaluation for the FBN1 gene was carried on 32 MFS patients. Gene mutation (n = 15, 47%) was classified as mild when the mutation resulted in nearly normally functioning protein, while mutations resulting in abnormally function protein were considered to be severe (n = 17, 53%). All patients and controls underwent 3D-STE for evaluation of LV function by an echocardiographer blinded to the results of the genetic testing. Compared to controls, MFS patients had significantly lower 3D-STE derived LV ejection fraction (EF, 57.43±7.51 vs. 62.69±4.76%, p = 0.0001), global LV longitudinal strain (LS, 14.85±2.89 vs. 17.90±2.01%, p = 0.0001), global LV circumferential strain (CS, 13.93±2.81 vs. 16.82±2.17%, p = 0.0001) and global LV area strain (AS, 25.76±4.43 vs. 30.51±2.61%, p = 0.0001). Apart from the global LV LS all these parameters were significantly lower in patients with severe gene mutation than in those with mild mutation (p<0.05). In the multivariate linear regression analysis only the type of mutation had a significant influence on the 3D-STE derived LVEF (p = 0.017), global CS (p = 0.005) and global AS (p = 0.03).

Conclusions

In asymptomatic MFS patients latent LV dysfunction can be detected using 3D STE. The LV dysfunction is mainly related to the severity of gene mutation, suggesting possible primary cardiomyopathy in MFS patients.     

Distance measures and optimization spaces in quantitative fatty acid signature analysis

Quantitative fatty acid signature analysis has become an important method of diet estimation in ecology, especially marine ecology. Controlled feeding trials to validate the method and estimate the calibration coefficients necessary to account for differential metabolism of individual fatty acids have been conducted with several species from diverse taxa. However, research into potential refinements of the estimation method has been limited. We compared the performance of the original method of estimating diet composition with that of five variants based on different combinations of distance measures and calibration‐coefficient transformations between prey and predator fatty acid signature spaces. Fatty acid signatures of pseudopredators were constructed using known diet mixtures of two prey data sets previously used to estimate the diets of polar bears Ursus maritimus and gray seals Halichoerus grypus, and their diets were then estimated using all six variants. In addition, previously published diets of Chukchi Sea polar bears were re‐estimated using all six methods. Our findings reveal that the selection of an estimation method can meaningfully influence estimates of diet composition. Among the pseudopredator results, which allowed evaluation of bias and precision, differences in estimator performance were rarely large, and no one estimator was universally preferred, although estimators based on the Aitchison distance measure tended to have modestly superior properties compared to estimators based on the Kullback–Leibler distance measure. However, greater differences were observed among estimated polar bear diets, most likely due to differential estimator sensitivity to assumption violations. Our results, particularly the polar bear example, suggest that additional research into estimator performance and model diagnostics is warranted.     

In Vivo Volatile Organic Compound Signatures of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests.     

Cardiovascular autonomic neuropathy in spontaneously diabetic rats with and without application of EGb 761

Cardiovascular autonomic neuropathy causes abnormalities in the diabetic heart with various clinical sequelae, including exercise intolerance, arrhythmias and painless myocardial infarction. Little is known about (ultra)structural alterations of the myocardial nervous network. On the assumption that this diabetes-specific neuropathy develops due to permanently increased oxidative stress by liberation of oxygen-free radicals, adjuvant application of antioxidative therapeutics appears promising in preventing or delaying long-term diabetic complications. We have investigated the effects of Ginkgo biloba extract (EGb 761), a radical scavenger, against diabetes-induced myocardial nervous damage in spontaneously diabetic BioBreeding/Ottawa Karlsburg (BB/OK) rats. Morphological and morphometric parameters were evaluated by electron microscopy. We used immunohistochemistry to investigate protein expression of protein gene product 9.5, S100 protein, and thyroxin hydroxylase as a neuronal marker. Alterations of cardiac sympathetic activity were measured using the in vivo 123I-metaiodobenzyl-guanidine imaging, and the immunofluorescent labeling of beta1-adrenergic receptors and adenylate cyclase. Our results revealed that A) Diabetes results in slight to moderate ultrastructural alterations (hydrops, disintegration of substructure) of autonomic nerve fibers and related Schwann cells in untreated BB diabetic rats; B) Cardiac sympathetic integrity and activity is impaired due to alterations in the presynaptic nerve terminals and the postsynaptic ?1-AR-AC coupling system; C) Pre-treatment of diabetic myocardium with EGb results in an improvement of most of these parameters compared to unprotected myocardium. In conclusion, EGb may act as a potent therapeutic adjuvant in diabetics with respect to cardiovascular autonomic neuropathy, which may contribute to the prevention of late complications in diabetes.     

Replication through an abasic DNA lesion: structural basis for adenine selectivity

Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A‐rule. In this study, we present X‐ray structures of a DNA polymerase caught while incorporating a nucleotide opposite an abasic site. We found that a functionally important tyrosine side chain directs for nucleotide incorporation rather than DNA. It fills the vacant space of the absent template nucleobase and thereby mimics a pyrimidine nucleobase directing for preferential purine incorporation opposite abasic residues because of enhanced geometric fit to the active site. This amino acid templating mechanism was corroborated by switching to pyrimidine specificity because of mutation of the templating tyrosine into tryptophan. The tyrosine is located in motif B and highly conserved throughout evolution from bacteria to humans indicating a general amino acid templating mechanism for bypass of non‐instructive lesions by DNA polymerases at least from this sequence family.     

Interactions Between Rhinanthus minor and Its Hosts: A Review of Water, Mineral Nutrient and Hormone Flows and Exchanges in the Hemiparasitic Association

At the ecological level, the effects of the facultative root hemiparasite Rhinanthus minor on the structure and functioning of its host communities are relative well described; yet until recently, the mechanistic basis for parasitic plant-driven community change and the physiological basis for the host-parasite interaction were poorly understood. Empirical incremental flow models, based on the increase in water, mineral nutrients, carbon assimilates or phytohormones between two defined time points, have been successfully employed to investigate the physiology of resource acquisition by- and distribution within host-parasitic plant associations. In this study we review the application of these empirical flow models to Rhinanthus-host associations showing the extent of and physiological basis of resource abstraction from the host and how this is profoundly influenced by soil nutrient status. We show that Rhinanthus primarily abstracts water and mineral nutrients via the apoplastic pathway through direct lumen-lumen connections with little resource acquisition via symplastic pathways. Nutrient status of the soil is shown to significantly influence the resource acquisition. We also investigate the hormonal regulation of resource acquisition by Rhinanthus showing pivotal roles for the key for the phytohormones abscisic acid (ABA) and cytokinins.