High-speed gel microelectrophoresis, a new and easy approach for detection of PCR-amplified microbial DNA from environmental and clinical samples in microgels using conventional equipment

AIMS: Microelectrophoresis allows the detection of DNA bands using minimal amounts of sample in a short time, but commonly requires the use of special equipment which is not available in all laboratories. This fact has limited the application of this technique in microbiology despite its advantages. In this work, we describe a new approach to perform gel microelectrophoresis, named high-speed gel microelectrophoresis (HSGME), and its application for rapid detection of bacteria, protozoa and viruses in clinical, vegetal and environmental samples. METHODS AND RESULTS: Aliquots of 0.4-1 microl of PCR product were loaded in 2 cm 1% agarose microgels and electrophoresed at high voltage (125 V cm(-1)) in conventional submarine horizontal mini-slabs. By using HSGME, single-DNA bands obtained after specific-PCR useful in diagnosis of different diseases caused by micro-organisms were detected in 5 min. CONCLUSIONS: HSGME is a rapid and easy procedure applicable to detection of microbial genes, which is carried out using conventional equipment and thus can be performed in any research and diagnostic laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: The performance of HSGME saves up to 90% time, material and energy costs, as well as laboratory hazardous wastes including carcinogenic agents used for visualizing DNA bands.     

Synthesis, isomer characterization, and anti-inflammatory properties of nitroarachidonate

Nitrated fatty acids (nitroalkenes) have been recently detected and quantified in cell membranes and human plasma. However, nitration of arachidonate (AA), that could redirect AA-dependent cell signaling pathways, has not been studied in detail. Herein, we synthesized and determined for the first time the isomer distribution of nitroarachidonate (AANO2) and demonstrate its ability to modulate inflammation. Synthesis of AANO2 was achieved by AA treatment with sodium nitrite in acidic conditions following HPLC separation. Mass spectrometry (MS) analysis showed the characteristic MS/MS transition of AANO2 (m/z 348/301). Moreover, the IR signal at 1378.3 cm(-1) and NMR studies confirmed the presence of mononitrated nitroalkenes. Positional isomer distribution was determined by NMR and MS fragmentation with lithium; four major isomers (9-, 12-, 14-, and 15-AANO2) were identified, which exhibited key anti-inflammatory properties. These include their ability to release biologically relevant amounts of nitric oxide, induce cGMP-dependent vasorelaxation, and down-regulate inducible nitric oxide synthase (NOS2) expression during macrophage activation, providing unique structural evidence and novel regulatory signaling properties of AANO2.     

Uterine and fetal asphyxia monitoring in parturient sows treated with oxytocin

Oxytocin is used to induce and control parturition, nevertheless, the increase of uterine contractions decreases blood flow and gaseous exchange through the womb predisposing to intra-partum mortality. The objective of the present study was to evaluate the effect of oxytocin on myometrial activity, fetal intrauterine hypoxia and postnatal asphyxia in sows during farrowing. Hybrid (n = 120) sows approaching the time of farrowing were randomly assigned in two groups of 60 animals each. Group I (G(1): control) was treated IM with saline solution and Group II (G(2)) was injected IM with oxytocin (1IU/6kg LW) as a single dose at birth of the first piglet. Both average number of myometrial contractions and intensity in G(2) were greater (P < 0.01) as compared with G(1). The mean of intra-partum stillbirths (IPS's) and those where fetal cardiac frequency (FCF) or heart beats, could not be detected after birth, were greater (P < 0.01) in G(2) as compared with G(1). The average decelerations of FCF known as dips II, which indicate severe hypoxia, was greater in G(2) (P < 0.01) as compared with that of G(1). There was a greater (P < 0.01) number of intra-partum stillbirths, stained with severe meconium in G(2) when compared with G(1). Oxytocin treatment increased (P < 0.01) the number of pigs born alive with ruptured umbilical cords and those with different grades of meconium staining on their skin. It was concluded that administration of oxytocin at the onset of parturition increased the myometrial activity, decreased fetal cardiac frequency, predisposed the rupture of umbilical cords and the degree of meconium staining, and increased intra-partum mortality.     

Effects of vanadium-containing compounds on membrane lipids and on microdomains used in receptor-mediated signaling

There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium (V) compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of V compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving V complexes such as [VO(2)(dipic)](-) (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V(10)O(28)(6-), V(10)), BMOV (bis(maltolato)oxovanadium(IV)), and [VO(saltris)](2) (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between V-containing compounds and model lipid systems, an evaluation of the effects of V compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of V-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms.     

Quaternary structure of pig liver phosphofructokinase

The quaternary structure of phosphofructokinase from pig liver has been studied by electron microscopy. Particles ranging in size from tetramers to long flexible chains of tetramers were commonly observed. Phosphofructokinase tetramers are square planar and approximately 110 A on a side; individual subunits are roughly spherical, with a mean radius of 28 A. Chains are formed by end-to-end association of tetramers rather than by tetramer stacking. The geometry of association implies that phosphofructokinase tetramers possess D2 symmetry, with distinct isologous bonding domains for dimer, tetramer, and chain formation.     

Neuronal typology of the thalamic area triangularis of Gallotia galloti (reptilia,sauria)

In a Golgi study of the area triangularis (AT), a rostral nucleus of the ventral thalamus of Gallotia galloti, we have identified four major neuronal types on the basis of their morphological characteristics: medium-sized fusiforms with two processes, medium-sized fusiforms with three or four processes, small bipolars, and small and medium-sized multipolars. These neurons are characterized by a simple morphology and radial arrangement. Cell size varies from small to medium, and all axons project laterally. These characteristics distinguish AT neurons from those of neighboring nuclei. In addition, we found some evidence of differential topographic distribution of each neuronal type within the nucleus. Medium-sized fusiform neurons with two processes are located in the most ventral part, where they constitute the ventral nuclear limit. Small multipolar neurons prevail in the dorsal and ventromedial parts, and in the rest of the nucleus medium-sized neurons, including both fusiform with three or four processes and multipolar types, are normally found. Finally, we discuss a putative homology of the reptilian AT with a part of the mammalian zona incerta.