Effects of the ΔF508 mutation on the structure,function, and folding of the first nucleotide-binding domain of CFTR

The fatal autosomal recessive disease cystic fibrosis (CF) is caused by mutations in the gene which encodes the cystic fibrosis transmembrane conductance regulator (CFTR). Many of these disease-causing mutations, including the deletion of F508 (F508) which accounts for approximately 70% of the disease alleles, occur in one of the two consensus nucleotide binding sequences. Peptide studies have directly demonstrated that the N-terminal nucleotide binding sequences bind adenine nucleotides. Structurally, circular dichroism spectropolarimetry indicates that this region of CFTR assumes a -stranded structure in solution. The F508 mutation causes a diminution in the amount of -stranded structure and a concomitant increase in the amount of random coil structure present, indicating that either the mutant peptide has a different native structure or that the conformational equilibrium is shifted toward a more disordered form. Furthermore, the mutant peptide is more sensitive to denaturation, indicating that F508 is a stability, or protein-folding mutant. Here we review these results and discuss their implications for interpreting the behavior of F508in situ and for the rational design of new CF drugs.     

Effects of 50 Hz electromagnetic fields on the histology, apoptosis, and expression of c-Fos and β-catenin on the livers of preincubated white Leghorn chicken embryos

Reports have demonstrated occurrences of abnormalities in the early stages of chicken embryonic development due to the exposure to electromagnetic fields (EMFs). This article was designed to investigate the effects of sinusoidal EMF on the histopathology, apoptosis, and expressions of c-Fos and β-Catenin genes of the livers of preincubated White Leghorn chicken embryos, based on our published experiments. 300 healthy, fresh fertilized eggs were divided into control (n?=?70), sham (n?=?70), and four experimental (1-4,days 13, 14, 5, and 19, n?=?40) groups. Experimental eggs were exposed to the most effective intensity in a coil with 7.32?mT density, and sham groups were also located in the same coil with no exposure, both for 24?h before incubation. Control, sham, and experimental groups were then incubated in an incubator (37°C, humidity 60%) for 13,14,15, and 19 days. Livers of 13-15 and 19 day-old chicken embryos were removed by C-section and fixed in formalin (10%), stained with Hematoxylin-Eosin and TUNEL for histopathological and apoptosis studies. Others were used for investigating c-Fos and β-Catenin expressions, using RT-PCR. Results showed extensive hemorrhages all over the chicken embryos' bodies and livers, more lymphoid tissues, disturbed parenchymal tissues, sinusoid denaturation, vesiculizad cytoplasm, an increase in the number of apoptotic cells, and a decrease on the levels of expressions of c-Fos and β-Catenin genes in experimental groups of 1-4, comparing control and sham groups.     

Effects of 50 Hz electromagnetic fields on the histology,apoptosis, and expression of c-Fos and β-Catenin on the livers of preincubated white leghorn chicken embryos

Reports have demonstrated occurrences of abnormalities in the early stages of chicken embryonic development due to the exposure to electromagnetic fields (EMFs). This article was designed to investigate the effects of sinusoidal EMF on the histopathology, apoptosis, and expressions of c-Fos and β-Catenin genes of the livers of preincubated White Leghorn chicken embryos, based on our published experiments. 300 healthy, fresh fertilized eggs were divided into control (n = 70), sham (n = 70), and four experimental (1–4,days 13, 14, 5, and 19, n = 40) groups. Experimental eggs were exposed to the most effective intensity in a coil with 7.32 mT density, and sham groups were also located in the same coil with no exposure, both for 24 h before incubation. Control, sham, and experimental groups were then incubated in an incubator (37°C, humidity 60%) for 13,14,15, and 19 days. Livers of 13–15 and 19 day-old chicken embryos were removed by C-section and fixed in formalin (10%), stained with Hematoxylin-Eosin and TUNEL for histopathological and apoptosis studies. Others were used for investigating c-Fos and β-Catenin expressions, using RT-PCR. Results showed extensive hemorrhages all over the chicken embryos' bodies and livers, more lymphoid tissues, disturbed parenchymal tissues, sinusoid denaturation, vesiculizad cytoplasm, an increase in the number of apoptotic cells, and a decrease on the levels of expressions of c-Fos and β-Catenin genes in experimental groups of 1–4, comparing control and sham groups.     

Effects of Eution Conditions on the Separation of Calpastatin, μ- and m-Calpain on DEAE-Sephacel Chromatography

A rapid stepwise measurement for the activities of calpastatin and μ- and m-calpains was developed by using 2-stage elution at pH 8.5 and then 7.0. The activities of calpastatin, μ-calpain and m-calpain can be rapidly assayed following the separation on DEAE-Sephacel chromatography by a 2 stage elution with 90 mM NaCl (pH 8.5), and then by 200 and 300 mM NaCl in elution buffer (pH 7.0). No significant differences in the recovery of these proteinases and inhibitor was observed between stepwise gradient and linear gradient methods.     

Effects of creatine and its analog, β-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.     

Effect of Dephosphorylation on the Self-association and the Precipitation of β-Casein

The pyrolyzate of the nondialyzable melanoidin prepared from glucose-ammonia reaction system (kept in pH 5.3~6.0 during the reaction) was fractionated to volatile fraction and nonvolatile fraction. Among the volatile components, two pyridines and four alkylpyrazines were identified. On the other hand, one imidazole compound and two β-hydroxypyridines isolated from the nonvolatile fraction were identified as 4(5)-methylimidazole, 3-hydroxypyridine and 2-methyl-5-hydroxypyridine, respectively. It is inferred that these compounds are not produced by the fission of the main skeleton in the melanoidin molecule, but formed by pyrolysis of the heterocyclic compounds present as a small moiety in the melanoidin.     

Microbial diversity of soils on the banks of the Solimões and Negro rivers, state of Amazonas, Brazil

Analysis of bacterial diversity in soils along the banks of the Solimões and Negro rivers, state of Amazonas, Brazil, was by partial sequencing of the genes codifying the rDNA16S region. Diversity of operational taxonomic units (OTU) and of the divergent sequences obtained were applied in comparative analysis of microbiological diversity in the two environments, based on richness estimators and OTU diversity indices. The higher OTU diversity in the Solimões was based on the higher number of parameters that evoke this. The interaction between the nucleotide sequences of bacteria inhabiting the two riverine environments indicated that the two microrganism communities are similar in composition.     

Differential effects of d-amphetamine, β-phenylethylamine,cocaine and methylphenidate on the rate of dopamine synthesis in terminals of nigrostriatal and mesolimbic neurons and on the efflux of dopamine metabolites into cerebroventricular perfusates of rats

The $\text{in}$$\text{vivo}$ of four psychomotor stimulants (d-amphetamine, β-phenylethylamine, cocaine and methylphenidate) were determined on: 1) the rate of dopamine (DA) synthesis, as measured by the accumulation of dihydroxyphenylalanine (DOPA) after aromatic L-amino acid decarboxylase inhibition, in the striatum (terminals of nigrostriatal neurons) and in the nucleus accumbens and olfactory tubercle (terminals of mesolimbic neurons) and 2) the efflux of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) into cerebroventricular perfusates of conscious, freely-moving rats. d-Amphetamine and β-phenylethylamine produced biphasic responses with lower doses of each drug increasing both the efflux of DOPAC and the rate of DA synthesis in the striatum. Higher doses of each drug either had no effect or actually decreased the efflux of DOPAC and also decreased the rate of DA synthesis in the striatum. Higher doses of each drug either had no effect only decreased the efflux of DOPAC and the rate of DA synthesis in the striatum. The effects of the drugs on the rate of DA synthesis in the nucleus accumbens and olfactory tubercle were similar to, but less pronounced than those seen in the striatum. These results are consistent with the following suggestions: 1) low doses of d-amphetamine and β-phenylethylamine facilitate the neuronal release of DA while higher doses of both drugs facilitate release and inhibit neuronal reuptake of the amine, and 2) cocaine and methylphenidate preferentially block the neuronal reuptake of DA.     

Impact of the spatial scale of grass–legume mixtures on sheep grazing behaviour,preference and intake,and subsequent effects on pasture

The benefits of using white clover in pastures have been widely recognised for many years. However, clover is perceived as being unreliable because of its typically low content, which is spatially and temporally variable, in mixed pastures. One proposed solution to increase the proportion of clover in the diet of grazing animals and composition in the pasture is to spatially separate clover from grass within the same field. In a field experiment ryegrass and white clover were grown in fine mixtures, and in pure alternating strips of ryegrass and clover of 1.5 m, 3 m or 18 m width within a field. Pastures were grazed for two grazing periods of 9 and 12 weeks, and measurements of sward surface height (SSH), herbage mass and composition and clover morphology were taken. Grazing behaviour was also observed. Results showed that spatial separation in the long term, when compared with a fine mixture, increased clover availability (18% to 30% v. 9%, based on standing dry matter) and was not grazed to extinction. Ewes maintained their preference for clover throughout the experiment (selection coefficient 2 to 5), which resulted in a reduction in the SSH of clover in monocultures to <3 cm and significant changes to the morphology of clover (smaller leaves, shorter petioles and thicker stolon), at the expense of maximising their intake. Spatial separation in the short term may therefore allow grazing animals to select their preferred diet; however, in the long term in continuously grazed pasture, their preference for clover depletes its availability.     

Impact of prehistoric settlements on the Cladocera in the sediments of Lakes Suszek,Bł\cedowo,and Skrzetuszewskie

An analysis of Cladocera has been made on sediments from the three Polish lakes Suszek, B\cedowo, and Skrzetuszewskie. The analysed sediments are gyttja from the Subboreal and Subatlantic periods, during which the lakes were subject variously to eutrophication. The eutrophication process has been analysed from the frequency of Cladocera, particularly the index species, and has been correlated with climatic and settlement changes. It was found that in Suszek lake, in which no significant human activity had taken place (palynologically documented), the eutrophication must have been slow and gradual. On the contrary, in the two remaining lakes, in which the human influence was more intense, the eutrophication process underwent intensive changes and coincided somewhat with settlement fluctuations.     

The Relative Significance of Host–Habitat,Depth, and Geography on the Ecology,Endemism, and Speciation of Coral Endosymbionts in the Genus Symbiodinium

Dinoflagellates in the genus Symbiodinium are among the most abundant and important group of eukaryotic microbes found in coral reef ecosystems. Recent analyses conducted on various host cnidarians indicated that Symbiodinium assemblages in the Caribbean Sea are genetically and ecologically diverse. In order to further characterize this diversity and identify processes important to its origins, samples from six orders of Cnidaria comprising 45 genera were collected from reef habitats around Barbados (eastern Caribbean) and from the Mesoamerican barrier reef off the coast of Belize (western Caribbean). Fingerprinting of the ribosomal internal transcribed spacer 2 identified 62 genetically different Symbiodinium. Additional analyses of clade B Symbiodinium using microsatellite flanker sequences unequivocally characterized divergent lineages, or “species,” within what was previously thought to be a single entity (B1 or B184). In contrast to the Indo-Pacific where host-generalist symbionts dominate many coral communities, partner specificity in the Caribbean is relatively high and is influenced little by the host’s apparent mode of symbiont acquisition. Habitat depth (ambient light) and geographic isolation appeared to influence the bathymetric zonation and regional distribution for most of the Symbiodinium spp. characterized. Approximately 80% of Symbiodinium types were endemic to either the eastern or western Caribbean and 40–50% were distributed to compatible hosts living in shallow, high-irradiance, or deep, low-irradiance environments. These ecologic, geographic, and phylogenetic patterns indicate that most of the present Symbiodinium diversity probably originated from adaptive radiations driven by ecological specialization in separate Caribbean regions during the Pliocene and Pleistocene periods.     

The effects of ambient temperature,nature and temperature of the floor and radiant heat on the metabolic rate of the new—born pig

Using a closed circuit technique, the oxygen consumption of 108 piglets below one week of age have been measured while modifying the physical attributes of the thermal environment. Moving the piglets onto a wooden floor from concrete reduced the oxygen consumption rate from 37.8 ml to 34.5 ml/kg. min) at 10°C, and from 33.7 to 31.4 ml/(kg. min) on a rubber floor at the same temperature. Oxygen consumption was also significantly reduced when the piglets were placed on a straw floor, were supplied with radiant heat and when the floor temperature was raised independently of chamber temperature. It is concluded that the provision of supplementary heating either in the floor or by overhead radiation can greatly reduce the effects of a cold environment on the new-born pig and so enhance the animal's viability.

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Zusammenfassung Der Sauerstoff-Umsatz von 108 Ferkeln, die jünger als eine Woche waren, wurde unter wechselnden Bedingungen der thermischen Umwelt gemessen. Das Verlegen der Ferkel von einem Zement- auf einen Holzboden führte zur Senkung des 0₂-Umsatzes von 37,8 auf 34,5 ml O₂/kg. min) und von 33,7 auf 31,4 ml O₂/(kg. min) bei 10°C auf einem Gummiboden. Auf einem strohbedecktem Boden war der O₂-Umsatz ebenfalls vermindert. Den gleichen Effekt hatte die Anwendung von Strahlungswärme oder die Erhöhung der Bodentemperatur. Danach kann zusätzliche Erwärmung vom Boden oder von der Decke die Kälteeinwirkung auf das Ferkel aus der Umgebung stark reduzieren und die Lebensfähigkeit der Tiere erhöhen.

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Resume Au moyen d'un appareil à circuit fermé, on a mesuré la consommation en oxygène le 108 porcelets âgés de moins d'une semaine soumis à divers régimes thermiques et cela dans une température ambiante constante. En transférant les porcelets d'un sol de béton à un plancher de bois, leur consommation d'oxygène a passé de 37,8 à 34,5 ml/(kg. min), et de 33,7 à 31,4 ml/(kg. min) en passant du béton à un sol recouvert de caoutchouc. La consommation d'oxygène a également été réduite de façon significative pour un sol recouvert de paille. Il en est allé de même pour des lampes à infra-rouge et si le sol était chauffé indépendamment du local. On en conclut qu'un chauffage supplémentaire soit dans le sol soit par rayonnement peut réduire sensiblement les effets néfastes d'une ambiance froide sur lesporcelets nouveau-nés et rehausser leurs chances de survie.

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Presented during the Fifth International Biometeorological Congress, 1–6 September 1969, Montreux, Switzerland.     

Kinetic and thermodynamic analysis of the inhibitory effects of maltose,glucose, and related carbohydrates on wheat β-amylase

Inhibition of wheat β-amylase (WBA) by glucose and maltose was studied by kinetics and thermodynamics. The inhibitory effects of fructose, difructose, sucrose, trehalose, cellobiose, acarbose, and 1-deoxynojirimycin on WBA were also evaluated. The half maximal inhibitory concentrations (IC₅₀) of acarbose, maltose and glucose were 0.06 ± 0.01 M, 0.22 ± 0.09 M, and 1.41 ± 0.17 M, respectively. The inhibitor constant (K_i) and the thermodynamic parameters such as changes in Gibbs energy (ΔG), enthalpy (ΔH), and entropy (ΔS) of the dissociation reactions of the WBA-glucose and WBA-maltose complexes were temperature and pH-dependent. The dissociation reactions were endothermic and enthalpy-driven. Both glucose and maltose behaved as competitive inhibitors at pH 3.0 and 5.4 at a temperature of 25 °C with respective K_i values of 0.33 ± 0.02 M and 0.12 ± 0.03 M. In contrast, both sugars exhibited uncompetitive inhibition at pH 9 at a temperature of 25 °C with K_i values of 0.21 ± 0.03 M for glucose and 0.11 ± 0.04 M for maltose. The pH-dependence of the inhibition type and K_i values indicate that the ionizing groups of WBA influence drastically the interaction with these carbohydrates. This evidence enables us to consider temperature and pH in the WBA-catalyzed hydrolysis to manipulate the inhibition by end-product, maltose, and even by glucose.     

Effect of experimental infection with Listeria monocytogenes on the development of pregnancy and on concentrations of progesterone,oestrone sulphate and 15-ketodihydro-PGF2α in the goat

The effect of Listeria monocytogenes infection on hormone levels in pregnant goats was studied. Four goats (Group I) received an intravenous inoculation of a bacterial culture (Type 1) on Days 69–77 and another four goats (Group II) received a similar inoculation on Days 105–106 of gestation. Five non-inoculated goats were used as controls. Plasma was analysed for progesterone, oestrone sulphate and 15-ketodihydro-PGF_2α. The status of the foetus was followed using real-time ultrasonography.Three of the four goats in Group I aborted 8–10 days after inoculation. The fourth goat gave birth to a normal live kid at term. The three goats which aborted showed clinical signs of disease in connection with abortion. In Group II, all goats aborted after 9–11 days. All the goats showed clinical symptoms of disease from a few days after inoculation and the symptoms continued until abortion. The clinical symptoms of disease were more pronounced in Group II than in Group I. L. monocytogenes was isolated from all aborted foetuses. None of the control goats aborted.Ultrasound examination revealed foetal death either immediately before or up to 2 days before abortion. Mummification had begun in the foetus that had been dead for 2 days before expulsion.In comparison with pre-inoculation plasma levels in Group I, a significant decrease (P < 0.05) in progesterone levels and an increase in 15-ketodihydro-PGF_2α levels were observed from Days 4 and 6 after inoculation, respectively. In Group II, a significant decrease (P < 0.05) in progesterone levels and an increase in 15-ketodihydro-PGF_2α levels in plasma were observed from Days 8 and 6, respectively. The oestrone sulphate levels decreased slightly in the inoculated goats a few days before abortion.The pattern of changes in levels around abortion was similar to the pattern present in the control animals around parturition. However, oestrone sulphate levels did not increase in the inoculated groups before abortion in contrast to goats which delivered healthy kids. The changes in levels of 15-ketodihydro-PGF_2α in goats that aborted indicated that the endocrine foetal-placental function was disturbed, which was most likely due to the establishment and development of L. monocytogenes in the placenta and foetus.     

Effect of γ-hexachloran and sevin on the survival of the Black Sea mussel,Mytilus galloprovincialis Lam

Some observations on the effect of -hexachloran and sevin on the survival of two size groups of the Black Sea mussel, Mytilus galloprovincialis Lam. were made. The results of the present study are compared with those made earlier on the same species and on fishes.     

Effect of the Support Size on the Properties of β-Galactosidase Immobilized on Chitosan: Advantages and Disadvantages of Macro and Nanoparticles

The effect of the support size on the properties of enzyme immobilization was investigated by using chitosan macroparticles and nanoparticles. They were prepared by precipitation and ionotropic gelation, respectively, and were characterized by Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), transmission electron microscopy (TEM), light scattering analysis (LSA), and N(2) adsorption-desorption isotherms. β-Galactosidase was used as a model enzyme. It was found that the different sizes and porosities of the particles modify the enzymatic load, activity, and thermal stability of the immobilized biocatalysts. The highest activity was shown by the enzyme immobilized on nanoparticles when 204.2 mg protein·(g dry support)(-1) were attached. On the other hand, the same biocatalysts presented lower thermal stability than macroparticles. β-Galactosidase immobilized on chitosan macro and nanoparticles exhibited excellent operational stability at 37 °C, because it was still able to hydrolyze 83.2 and 75.93% of lactose, respectively, after 50 cycles of reuse.     

Effect of Silt, Water and Periphyton Quality on Survival and Growth of the Mayfly Heptagenia Sulphurea

The herbivorous mayfly Heptagenia sulphurea is characteristic of rivers with stony bottoms. Records from the 20th century showed that this species had disappeared from the Common Meuse in the Netherlands, probably due to river regulation or changes in water quality. A field survey in 2003 showed that H. sulphurea was present in the Geul tributary, approximately 300 m upstream of its confluence with the Common Meuse. H. sulphurea has not recolonized the Common Meuse despite improvements in water quality over the last decades. Concentration of suspended sediments in the River Meuse, however, is still high, much higher than in the beginning of the 20th century. The presence of a silt layer may limit the return of H. sulphurea in this river by reducing availability and quality of its food. The prime objective of this study was to investigate the impact of silt on survival and growth of H. sulphurea in a laboratory experiment. Furthermore, the impact of water and periphyton quality from the Common Meuse on survival and growth of this mayfly was also investigated. Results showed that neither water quality nor cultured periphyton from the Common Meuse reduced survival and growth of H. sulphurea. The presence of a silt layer resulted in a significantly lower growth rate of the mayfly larvae. It is concluded that the silt layer reduces the accessibility of the food. Covering of food is possibly one of the main factors limiting the recolonization of H. sulphurea and probably other benthic grazers in the Common Meuse.     

Effects of wild-type and α-tocopherol-enriched transgenic Brassica juncea on the components of xenobiotic metabolism, antioxidant status, and oxidative stress in the liver of mice

Alpha (α)-tocopherol is the most biologically active and preferentially retained form of vitamin E in the human body and is known for its antioxidant and gene regulatory functions. Its increased intake is implicated in protection against diseases that involve an oxidative stress component. We have evaluated the chemopreventive potential of a diet supplemented with natural α-tocopherol-enriched transgenic (TR) Brassica juncea seeds. The modulation of phase I and phase II xenobiotic metabolism and of antioxidative enzymes was compared in the livers of mice fed on a control diet or on a diet supplemented with 2, 4, and 6 % (w/w) of wild-type (WT) or TR seeds. A dose-dependent increase in the specific activities of these enzymes was observed in those animals fed on diet supplemented with TR seeds. In comparison, an increase in the specific activities of antioxidative enzymes was substantial only at higher doses of WT seeds. Consequently, oxidative stress measured in terms of lipid peroxidation and lactate dehydrogenase activity was found to be lower in the case of mice fed with the supplemented diet. However, the chemopreventive potential of TR seeds was more pronounced than that of WT seeds. This study demonstrates the feasibility of fortifying diets with natural α-tocopherol for chemopreventive benefits by means of transgenic manipulation of a commonly used oilseed crop.     

Effects of nandrolone and resistance training on the blood pressure,cardiac electrophysiology,and expression of atrial β-adrenergic receptors

AimsThis study was performed to assess isolated and combined effects of nandrolone and resistance training on the blood pressure, cardiac electrophysiology, and expression of the β₁- and β₂-adrenergic receptors in the heart of rats.Main methodsWistar rats were randomly divided into four groups and submitted to a 6-week treatment with nandrolone and/or resistance training. Cardiac hypertrophy was accessed by the ratio of heart weight to the final body weight. Blood pressure was determined by a computerized tail-cuff system. Electrocardiography analyses were performed. Western blotting was used to access the protein levels of the β₁- and β₂-adrenergic receptors in the right atrium and left ventricle.Key findingsBoth resistance training and nandrolone induced cardiac hypertrophy. Nandrolone increased systolic blood pressure depending on the treatment time. Resistance training decreased systolic, diastolic and mean arterial blood pressure, as well as induced resting bradycardia. Nandrolone prolonged the QTc interval for both trained and non-trained groups when they were compared to their respective vehicle-treated one. Nandrolone increased the expression of β₁- and β₂-adrenergic receptors in the right atrium for both trained and non-trained groups when they were compared to their respective vehicle-treated one.SignificanceThis study indicated that nandrolone, associated or not with resistance training increases blood pressure depending on the treatment time, induces prolongation of the QTc interval, and increases the expression of β₁- and β₂-adrenergic receptors in the cardiac right atrium, but not in the left ventricle.