Local dynamics of DNA probed with optical absorption spectroscopy of bound ethidium bromide.

We have studied the local dynamics of calf thymus double-helical DNA by means of an "optical labeling" technique. The study has been performed by measuring the visible absorption band of the cationic dye ethidium bromide, both free in solution and bound to DNA, in the temperature interval 360-30 K and in two different solvent conditions. The temperature dependence of the absorption line shape has been analyzed within the framework of the vibronic coupling theory, to extract information on the dynamic properties of the system; comparison of the thermal behavior of the absorption band of free and DNA-bound ethidium bromide gave information on the local dynamics of the double helix in the proximity of the chromophore. For the dye free in solution, large spectral heterogeneity and coupling to a "bath" of low-frequency (soft) modes is observed; moreover, anharmonic motions become evident at suitably high temperatures. The average frequency of the soft modes and the amplitude of anharmonic motions depend upon solvent composition. For the DNA-bound dye, at low temperatures, heterogeneity is decreased, the average frequency of the soft modes is increased, and anharmonic motions are hindered. However, a new dynamic regime characterized by a large increase in anharmonic motions is observed at temperatures higher than approximately 280 K. The DNA double helix therefore appears to provide, at low temperatures, a rather rigid environment for the bound chromophore, in which conformational heterogeneity is reduced and low-frequency motions (both harmonic vibrations and anharmonic contributions) are hindered. The system becomes anharmonic at approximately 180 K; however, above approximately 280 K, anharmonicity starts to increase much more rapidly than for the dye free in solution; this can be attributed to the onset of wobbling of the dye in its intercalation site, which is likely connected with the onset of (functionally relevant) DNA motions, involving local opening/unwinding of the double helix. As shown by parallel measurements of the melting curves, these motions precede the melting of the double helix and depend upon solvent composition much more than does the melting itself.     

Adhesiveness and surface patterns of eggs in neotropical freshwater teleosts

Eggs of 23 Characiformes and eight Siluriformes, belonging to nine families with diverse reproductive behaviour, were ultrastructurally analysed. The migratory species exhibited non-adhesive eggs, whereas, most of the sedentary species presented some degree of egg adhesiveness. Among the Characiformes, non-adhesive eggs showed zona radiata with pore-canals or a fibrillar net at the surface; weakly adhesive eggs presented only zona radiata with pore-canals while adhesive eggs exhibited zona radiata with apparatus like globules, filaments, villi or honeycomb-like pores depending on the systematic group. The 'jelly' coat is strongly related to the Siluriformes eggs apparently without relationship with adhesiveness. A micropylar disc was present in adhesive eggs of a few species of both Characiformes and Siluriformes. Some patterns were characteristic of the animal pole, others of the vegetal pole, and others were common to both poles. The radial ridges converging to the micropyle in Astyanax bimaculatus lacustris appear to be related to fertilization. In general, egg surface structures in the Characiformes varied according to the genus, whereas all Siluriformes showed a similar egg surface pattern, regardless of the group analysed. Multivariate analysis allowed the identification of eight clusters among the Characiformes and three among the Siluriformes showing relationships between reproductive style and egg characteristics. It is suggested that egg surface and adhesiveness may be related to reproductive patterns and to phylogenesis.     

Comparative oocyte morphology and early development in three species of trahiras from the São Francisco River basin, Brazil

Oocyte morphology, embryogenesis and early larval development were compared in Hoplerythrinus unitaeniatus , Hoplias lacerdae and Hoplias malabaricus (Characiformes: Erythrinidae) by macroscopical, histological, histochemical and ultrastructural analyses. The eggs of the three species were yellowish and adhesive, containing carboxyl and sulphate radicals in the glycoconjugates of the zona radiata. A complex surface arrangement was identified in oocytes of H. unitaeniatus and H. lacerdae , while H. malabaricus had a simple oocyte surface pattern. Lectin histochemistry revealed different carbohydrate terminal residues in cortical alveoli, outer zona radiata and follicular cells of the three species. At the animal pole, the oocyte surface topography surrounding the micropyle was species-specific. The micropylar cell was ConA-positive, suggesting the presence of carbohydrates with mannose/glucose terminal residues that could have a role during fertilization. The erythrinids exhibited a prolonged embryonic and larval development compared to other Characiformes, a reproductive strategy used for increasing offspring protection. Early development proceeded most rapidly in H. unitaeniatus , followed by H. malabaricus and then H. lacerdae , which could have more developed parental care behaviour. An adhesive organ composed of secretory prismatic cells protruding from the cephalic region of the three erythrinid larva allowed them to attach to one another during development. Reproductive behaviour and early developmental strategies were similar in the three species, but the oocyte surface morphology suggests a close relationship between H. unitaeniatus and H. lacerdae .     

Regulation of Glucokinase by Intracellular Calcium Levels in Pancreatic β Cells

Glucokinase (GCK) controls the rate of glucose metabolism in pancreatic β cells, and its activity is rate-limiting for insulin secretion. Posttranslational GCK activation can be stimulated through either G protein-coupled receptors or receptor tyrosine kinase signaling pathways, suggesting a common mechanism. Here we show that inhibiting Ca²⁺ release from the endoplasmic reticulum (ER) decouples GCK activation from receptor stimulation. Furthermore, pharmacological release of ER Ca²⁺ stimulates activation of a GCK optical biosensor and potentiates glucose metabolism, implicating rises in cytoplasmic Ca²⁺ as a critical regulatory mechanism. To explore the potential for glucose-stimulated GCK activation, the GCK biosensor was optimized using circularly permuted mCerulean3 proteins. This new sensor sensitively reports activation in response to insulin, glucagon-like peptide 1, and agents that raise cAMP levels. Transient, glucose-stimulated GCK activation was observed in βTC3 and MIN6 cells. An ER-localized channelrhodopsin was used to manipulate the cytoplasmic Ca²⁺ concentration in cells expressing the optimized FRET-GCK sensor. This permitted quantification of the relationship between cytoplasmic Ca²⁺ concentrations and GCK activation. Half-maximal activation of the FRET-GCK sensor was estimated to occur at ∼400 nm Ca²⁺. When expressed in islets, fluctuations in GCK activation were observed in response to glucose, and we estimated that posttranslational activation of GCK enhances glucose metabolism by ∼35%. These results suggest a mechanism for integrative control over GCK activation and, therefore, glucose metabolism and insulin secretion through regulation of cytoplasmic Ca²⁺ levels.     

Determination of new 2,3-benzodiazepines in rat plasma using high-performance liquid chromatography with ultraviolet detection

A method for the analysis of [1-(4-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one] (CFM-2) and its analogues CFM-3, CFM-4 and CFM-5 in rat plasma was developed. The 2,3-benzodiazepines (2,3-BZs) were extracted by liquid–liquid extraction and analyzed using high-performance liquid chromatography (HPLC) with ultraviolet detection (UV) at 240 nm. The method exhibited a large linear range from 0.05 to 2 μg/ml with an intra-assay accuracy for all studied compounds ranging from 92 to 105.5%; whereas the intra-assay precision ranged from 0.59 to 8.16% in rat plasma. The inter-assay accuracy of CFM-2, CFM-4 and their 3-methyl derivatives, CFM-3 and CFM-5 ranged from 92.2 to 107% and the inter-assay precision ranged from 2.17 to 11.9% in rat plasma. The lower limit of detection was 5.5 ng/ml for CFM-2, 6.5 ng/ml for CFM-3, 7 ng/ml for CFM-4 and 8.5 ng/ml for CFM-5 in rat plasma. The pharmacokinetic study demonstrated that 2,3-BZs achieved a peak plasma concentration between 45 and 75 min after drug administration. Moreover, we observed that plasma chromatograms of rats treated with CFM-3, CFM-4 and CFM-5, respectively, showed a peak consistent with CFM-2. Our study suggests that CFM-4, CFM-5 and CFM-3 are prodrugs of CFM-2, in which they are biotransformed in vivo via different metabolic pathways. In particular, CFM-2 has been proven to possess anticonvulsant activity in various models of seizures, acting as α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist.     

Antibody Immunodiversity: A Study on the Marked Specificity Difference Between Two Anti-Yeast Iso-1 Cytochrome c Monoclonal Antibodies Whose Epitopes Are Closely Related

Anti-yeast iso-1 cytochrome c (cyt. c) monoclonal antibodies 2-96-12 and 4-74-6 have closely related epitopes (antigenic determinants). However, while the specificity of 4-74-6 is stringent, 2-96-12 cross-reacts with many evolutionarily related cytochromes c. Such a marked difference in specificity of antibodies with overlapping epitopes may represent unique antibody immunodiversity. Thus, we constructed Fv fragment models consisting of the variable domains of the heavy and light chains of 2-96-12 and 4-74-6 and that of another anti-iso-1 cyt. c as a control to gain insight into the origin of this difference in specificity. Our models show that 4-74-6 and 2-96-12 contain five and two aromatic side chains, respectively, in or near the central area of the antigen-combining site. The side chains of Arg95H (heavy chain) in 2-96-12 and Arg91L (light chain) in 4-74-6 project toward the central area of the combining site in our model. Antigen docking to our Fv models, combined with previous immunological studies, suggests that iso-1 cyt. c Asp60 may interact with Arg95H in 2-96-12 and Arg91L in 4-74-6 and that both epitopes of 2-96-12 and 4-74-7 may include iso-1 cyt. c Leu58, Asp60, Asn62, and Asn63. The effect of the Arg95H to Lys mutation on the antigen binding is also in accord with our model. The difference in specificity may be partly explained by a greater degree of conformational flexibility in and around the central area of the combining site in 2-96-12 compared to 4-74-6 due to differences in aromatic side chain packing.     

Change of serum mitochondrial uncoupling protein 1 (UCP1) levels and daily rhythm of rectal and cutaneous temperatures in Equus caballus and Capra hyrcus

The aim of the present study was to monitor the daily rhythm of rectal and cutaneous temperatures together with the changes in the serum levels of mitochondrial uncoupling protein 1 (UCP1) in equine and goat species. On five gelding horses and five female goat rectal and cutaneous temperatures were recorded at 4 h intervals for a 48 h period. At the same time points blood samples were collected from each animal. Daily rhythms of rectal and cutaneous temperatures were observed in both species, in both day of monitoring. Mesor value of rectal temperature was statistically higher of about 4 °C than cutaneous temperature, and acrophase was postponed of about 2 h in both day of monitoring and in both species. UCP1 did not show daily rhythmicity in horses and goats. We can speculate that the thermogenesis due to thermal system is auxiliary to keeping the body temperature daily rhythm.