Preparation of subcellular fractions suitable for biochemical analyses from human subcutaneous adipose tissue obtained by needle biopsy. I. Isolation of mitochondria on a microscale

1.

1. A technique has been developed for the isolation of subcellular organelles on a microscale which can be used to examine small samples of tissue (10 to 40 mg). Separation is accomplished with a series of Millipore filters of appropriate pore size.     

Opposite effects of tumor necrosis factor and soluble fibronectin on junctional adhesion molecule-A in endothelial cells

Junctional adhesion molecule-A (JAM-A) regulates key inflammatory responses, such as edema formation and leukocyte transmigration. Although it has been reported that the inflammatory cytokine tumor necrosis factor (TNF) causes the disassembly of JAM-A from the intercellular junctions, the mechanism has not been elucidated fully. Here, we report that TNF enhances the solubility of JAM-A in Triton X-100 and increases the amount of Triton-soluble JAM-A dimers at the cell surface but does not change the total levels of cellular JAM-A. Thus we hypothesized that TNF causes the redistribution of JAM-A from the junctions to the cell surface and that junction disassembly is sufficient to account for JAM-A redistribution. Intriguingly, however, even after complete disassembly of the junctions (with EDTA and trypsin), higher levels of JAM-A are detectable at the cell surface (by FACS analysis) in cells that had been previously incubated in the presence of TNF than in its absence. Thus we propose that TNF causes not only the disassembly of JAM-A from the junctions and its subsequent redistribution to the cell surface but also its dispersal in such a way that JAM-A becomes more easily accessible to the antibodies used for FACS analysis. Finally, we evaluated whether soluble fibronectin might attenuate the effects of TNF on JAM-A, as some inflammatory conditions are associated with the depletion of plasma fibronectin. We found that fibronectin reduces the effect of TNF on the disassembly of JAM-A, but not on its dispersal, thus further stressing that disassembly and dispersal can be functionally dissociated.     

Complex segregation analysis of non-myoclonic idiopathic generalized epilepsy in families ascertained from probands affected with idiopathic epilepsy with tonic-clonic seizures in Antioquia,Colombia

In an attempt to identify the possible role of major genes, multifactorial inheritance, and cohort effects in the susceptibility to idiopathic epilepsy with generalized tonic-clonic seizures of the awakening type (GTCS), complex segregation analysis was performed in 196 nuclear families ascertained through affected probands with idiopathic epilepsy with GTCS belonging to the Paisa community of Antioquia (Colombia). Models postulating no transmission, single major locus (dominant and recessive) only, and multifactorial component only, were rejected. Since the codominant single major locus model could not be rejected and models that assign no major locus to transmission, no polygenic component to transmission, and no transmission of the major effect were rejected, complex segregation analysis suggested that a major autosomal codominant allele together with a multifactorial component (mixed model) best explained clustering of idiopathic epilepsy with GTCS in families of the Paisa community. The deficit of transmission of heterozygotes (0.17) is compatible with the existence of epistasis acting on a major gene whose frequency was estimated to be 0.0211. Its transmission variance accounts for 81% of the susceptibility to idiopathic epilepsy with GTCS. The complementary variance (19%) is due to the polygenic component. Received: 19 January 1996 / Revised: 11 March 1996     

Glutamate Release and Free Radical Production Following Brain Injury: Effects of Posttraumatic Hypothermia

Abstract: Posttraumatic hypothermia reduces the extent of neuronal damage in remote cortical and subcortical structures following traumatic brain injury (TBI). We evaluated whether excessive extracellular release of glutamate and generation of hydroxyl radicals are associated with remote traumatic injury, and whether posttraumatic hypothermia modulates these processes. Lateral fluid percussion was used to induce TBI in rats. The salicylate-trapping method was used in conjunction with microdialysis and HPLC to detect hydroxyl radicals by measurement of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA). Extracellular glutamate was measured from the same samples. Following trauma, brain temperature was maintained for 3 h at either 37 or 30°C. Sham-trauma animals were treated in an identical manner. In the normothermic group, TBI induced significant elevations in 2,3-DHBA (3.3-fold, p < 0.01), 2,5-DHBA (2.5-fold, p < 0.01), and glutamate (2.8-fold, p < 0.01) compared with controls. The levels of 2,3-DHBA and glutamate remained high for approximately 1 h after trauma, whereas levels of 2,5-DHBA remained high for the entire sampling period (4 h). Linear regression analysis revealed a significant positive correlation between integrated 2,3-DHBA and glutamate concentrations ( p < 0.05). Posttraumatic hypothermia resulted in suppression of both 2,3- and 2,5-DHBA elevations and glutamate release. The present data indicate that TBI is followed by prompt increases in both glutamate release and hydroxyl radical production from cortical regions adjacent to the impact site. The magnitude of glutamate release is correlated with the extent of the hydroxyl radical adduct, raising the possibility that the two responses are associated. Posttraumatic hypothermia blunts both responses, suggesting a mechanism by which hypothermia confers protection following TBI.     

A lyophilized aqueous extract of<Emphasis Type="Italic"> Maytenus ilicifolia</Emphasis> leaves inhibits histamine-mediated acid secretion in isolated frog gastric mucosa

Lyophilized aqueous extract of Maytenus ilicifolia leaves (LAEMIL) is commonly used in Brazilian folk medicine in the treatment of dyspepsia as well as gastric ulcers. We have investigated the effect and the possible mechanism of action of the LAEMIL on acid secretion in isolated frog gastric mucosa incubated in an Ussing chamber. It was observed that LAEMIL (7–28 mg%) as well as cimetidine (125–4,000 M), a well-known histamine H₂ receptor antagonist, decreased basal acid secretion in a concentration-dependent manner. Similarly to cimetidine (190 M), LAEMIL (21 mg%) also inhibited gastric acid secretion induced by increasing concentrations of histamine (50–800 M). The EC₅₀ values for histamine alone and histamine in the presence of LAEMIL or cimetidine were 94.6 M (71.1–125.9 M), 244.9 M (209.4–286.4 M) and 142.2 M (23.6–855.0 M), respectively. LAEMIL, histamine and cimetidine were effective on acid secretion only when added to the serosal surface of the mucosa. Furthermore, simultaneous addition of LAEMIL and cimetidine at concentrations, per se, ineffective, caused a 16% reduction in the basal acid secretion [from 8.3±0.3 to 6.9±0.2 Eq g^–1 (15 min)^–1, n=4]. Although effects such as inhibition of histamine biosynthesis and/or histamine release can not be ruled out, our data suggest that LAEMIL, like cimetidine, reduces acid secretion in the isolated frog gastric mucosa by antagonising histamine H₂ receptors.Abbreviations LAEMIL Lyophilized aqueous extract of Maytenus ilicifolia leaves - Hist Histamine - Cim Cimetidine     

Mitochondrial Creatine Kinase Binding to Phospholipid Monolayers Induces Cardiolipin Segregation

It is well established that the octameric mitochondrial form of creatine kinase (mtCK) binds to the outer face of the inner mitochondrial membrane mainly via electrostatic interactions with cardiolipin (CL). However, little is known about the consequences of these interactions on membrane and protein levels. Brewster angle microscopy investigations provide, for the first time to our knowledge, images indicating that mtCK binding induced cluster formation on CL monolayers. The thickness of the clusters (10-12 nm) corresponds to the theoretical height of the mtCK-CL complex. Protein insertion into a condensed CL film, together with monolayer stabilization after protein addition, was observed by means of differential capacity measurements. Polarization modulation infrared reflection-absorption spectroscopy showed that the mean orientation of α-helices within the protein shifted upon CL binding from 30° to 45° with respect to the interface plane, demonstrating protein domain movements. A comparison of data obtained with CL and phosphatidylcholine/phosphatidylethanolamine/CL (2:1:1) monolayers indicates that mtCK is able to selectively recruit CL molecules within the mixed monolayer, consolidating and changing the morphology of the interfacial film. Therefore, CL-rich domains induced by mtCK binding could modulate mitochondrial inner membrane morphology into a raft-like organization and influence essential steps of mitochondria-mediated apoptosis.     

Geographic isolation and long-distance gene flow influence the genetic structure of the blue fan palm Brahea armata (Arecaceae)

Journal of Plant Research - The formation of the Baja California Peninsula (BCP) has impacted the microevolutionary dynamics of different species in ways that depend on biological traits such as...     

Transition from exo- to endo- Cu absorption in CuSi(n) clusters: A Genetic Algorithms Density Functional Theory (DFT) Study

The characterization and prediction of the structures of metal silicon clusters is important for nanotechnology research because these clusters can be used as building blocks for nano devices, integrated circuits and solar cells. Several authors have postulated that there is a transition between exo to endo absorption of Cu in Si(n) clusters and showed that for n larger than 9 it is possible to find endohedral clusters. Unfortunately, no global searchers have confirmed this observation, which is based on local optimizations of plausible structures. Here we use parallel Genetic Algorithms (GA), as implemented in our MGAC software, directly coupled with DFT energy calculations to show that the global search of CuSi(n) cluster structures does not find endohedral clusters for n < 8 but finds them for n ≥ 10.     

Genetic and morphometric characterization of clones of Prosopis alba,Algarobia, selected for salt tolerance

Prosopis alba is an important Argentinean species with a great potential for the production of timber and nontimber products. Many studies showed high salt tolerance of this species, which allows it to be used in afforestation and reforestation of saline soils. In this study, we applied the morphometric technique to characterize 21 salt-tolerant clones (ST). Twenty of these clones were studied by inter-simple sequence repeat (ISSR) and simple sequence repeat (SSR), and their molecular patterns were compared with those of 22 individuals selected for salt sensitivity (SS). Most morphological traits revealed highly significant differences among ST clones, and four out of 11 characters showed high heritability. ISSR analysis allowed detecting 89 loci, 91 % of them variable. ST versus SS groups differ significantly from each other by the frequencies of 22 of these loci, from which 12 were significant at the matrix level. Analysis of six SSR loci for the same groups indicated that all of them were polymorphic at the 1 % criterion. Allelic frequencies of SSR also showed highly significant differences between SS and ST groups. Analysis of coancestry between individuals within SS and ST groups and between groups indicated that the molecular differentiation between them cannot be explained solely on relationship grounds. Molecular groupings based on ISSR and SSR showed consistency to each other, as supported by the highly significant coinertia in the distribution of individuals in principal component analysis scatterplots. This work is the first contribution which tends to associate molecular patterns with life history traits and morphological differences in Prosopis clones.