Enzymatic activity assay of D-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of D-hydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/time) generated. Microcalorimetric assays carried out at different temperatures provided the dependence of the catalytic rate constant on temperature. We show that ITC assay is a nondestructive method that allows the determination of the catalytic rate constant (kcat), Michaelis constant (KM), activation energy and activation Gibbs energy, enthalpy and entropy of this reaction.     

Cooperativity and flexibility of the protonmotive activity of mitochondrial respiratory chain

Functional and structural data are reviewed which provide evidence that proton pumping in cytochrome c oxidase is associated with extended allosteric cooperativity involving the four redox centers in the enzyme . Data are also summarized showing that the H+/e- stoichiometry for proton pumping in the cytochrome span of the mitochondrial respiratory chain is flexible. The DeltapH component of the bulk-phase membrane electrochemical proton gradient exerts a decoupling effect on the proton pump of both the bc1 complex and cytochrome c oxidase. A slip in the pumping efficiency of the latter is also caused by high electron pressure. The mechanistic and physiological implications of proton-pump slips are examined. The easiness with which bulk phase DeltapH causes, at least above a threshold level, decoupling of proton pumping indicates that for active oxidative phosphorylation efficient protonic coupling between redox complexes and ATP synthase takes place at the membrane surface, likely in cristae, without significant formation of delocalized DeltamuH+. A role of slips in modulating oxygen free radical production by the respiratory chain and the mitochondrial pathway of apoptosis is discussed.     

A safe haven for potential reproductive aggregations of the critically endangered Brazilian guitarfish (Pseudobatos horkelii)

Brazilian endemic batoid elasmobranch populations have declined dramatically in the past 40 years due to anthropic activities (e.g., overfishing). The Brazilian guitarfish, Pseudobatos horkelii, included in the IUCN red list of endangered species [Critically Endangered (CR)], has been captured as by-catch by trawling fishing boats to the edge of extinction. Despite governmental conservation initiatives, the species is still caught and commercialized along the Brazilian coast. In this study, the authors report three rare aggregation events for the Brazilian coast of P. horkelii, inside the only nearshore no-entry Brazilian marine protected area. Strategies for its protection are also discussed.     

An operative approach to correct CD spectra distortions due to absorption flattening

We discuss the influence of absorption flattening (AF) on CD spectra, aiming at finding a simple way to compensate the induced distortions. A simple algebraic formalism is proposed based on an unconventional measuring approach, which makes use of a commercial unit. Validation of the proposed compensation method for AF is conducted not only on the already studied CD Vis spectral region of a Co chiral complex with the use of an AF emulator, but also on the far UV CD spectra of poly-L-glutamic acid suspensions with different turbidity levels. Comparison with some correction factors proposed in the literature is presented.     

Myotonic dystrophytype 1 – report of non-24-h sleep-wake disorder with excessive daytime sleepiness

ABSTRACT

Myotonic dystrophy (MD) is a neuromuscular disease with myotonia, progressive weakness, and involvement of CNS, heart, and gastrointestinal system. Excessive daytime sleepiness (EDS) in myotonic dystrophy type 1 (MD1) is related to sleep breathing diseases, restless leg syndrome, periodic limb movements during sleep and narcoleptic-like phenotype. However, authors highlight a central dysfunction of sleep regulation.

We describe a 26-year-old, female, MD1 patient with EDS. Sleep diary/actigraphy evidenced two different circadian periods with values of 1442 and 1522 min. Agomelatine, 50 mg at night, was prescribed with improvement of the circadian rhythm and complaints of sleepiness.

The identification of unanticipated causes of EDS, such as circadian rhythm disorders permits an appropriated treatment. As we know, it is the first relate of non-24-h sleep-wake disorder in patient with MD1. Sleep diary and actigraphy could be good options to investigate sleep-wake cycle disorder in patients with MD and EDS.     

Carbohydrate metabolism and fruit quality are affected in frost-exposed Valencia orange fruit

Exposure of citrus fruit to frost often results in the development of freeze injury during their maturation in planta. This work was aimed to analyze changes in the biochemistry and enzymology of carbohydrate metabolism in freeze-injured orange fruit ( Citrus sinensis var. Valencia late) and the involvement of oxidative stress in frost damage. The activities of pyrophosphate-dependent phosphofructokinase, phosphoenolpyruvate carboxylase and several fermentative enzymes increased in frost-exposed (FE) fruit, while NADP-malic enzyme and the mitochondrial isoform of NAD-malate dehydrogenase showed a reduction in their activities. Western blot analysis indicated a correlation between activity levels and protein content. Respiration rate in whole fruit was reduced by 40%, whereas the flavedo showed a more pronounced decline (53%). Volatile compound (i.e. ethanol and acetaldehyde) content was significantly higher in FE fruit than in control, as was that of l -malate (three-fold). Additionally, FE fruit showed a marked decrease in the maturity index (24%) because of a higher titratable acidity (39%). Evidence is presented that oxidative stress is involved in freeze-induced damage of orange fruit, where oxidative damage to lipids and proteins, and a greater electrolyte leakage in the flavedo were also observed. The results suggest that freezing temperatures provoke a notable metabolic switch in citrus fruit toward a fermentative stage, resulting in low-quality fruits.     

Cliffs Used as Communal Roosts by Andean Condors Protect the Birds from Weather and Predators

The quality and availability of resources influence the geographical distribution of species. Social species need safe places to rest, meet, exchange information and obtain thermoregulatory benefits, but those places may also serve other important functions that have been overlooked in research. We use a large soaring bird that roosts communally in cliffs, the Andean condor (Vultur gryphus), as a model species to elucidate whether roost locations serve as a refuge from adverse weather conditions (climatic refuge hypothesis, CRH), and/or from predators or anthropogenic disturbances (threats refuge hypothesis, TRH). The CRH predicts that communal roosts will face in the opposite direction from where storms originate, and will be located in climatically stable, low precipitation areas. The TRH predicts that communal roosts will be large, poorly accessible cliffs, located far from human-made constructions. We surveyed cliffs used as communal roosts by condors in northwestern Patagonia, and compared them with alternative non-roosting cliffs to test these predictions at local and regional scales. We conclude that communal roosting places provide refuge against climate and disturbances such as, for instance, the threats of predators (including humans). Thus, it is not only the benefits gained from being aggregated per se, but the characteristics of the place selected for roosting that may both be essential for the survival of the species. This should be considered in management and conservation plans given the current scenario of global climate change and the increase in environmental disturbances.     

DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth

Background

Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before.

Methodology

We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa) using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker.

Conclusions

Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials.