Birth and evolutionary history of a human minisatellite

One of the most exciting challenges in human biology is the understanding of how our genome was constructed during evolution. Here we explore the evolutionary history of the low polymorphic human minisatellite MsH42 and its flanking sequences. We show that the evolutionary birth of MsH42 took place within an intron, early in primate lineage evolution, more than 40 MYA. Then, single base-pair changes and duplications/deletions of repeat blocks by mispairing were probably the main forces governing the generation of this minisatellite and its polymorphism throughout primate evolution. Moreover, we detected several phylogenetic footprints at both sides of MsH42. We believe that our findings will contribute to the understanding of low-variability minisatellite evolution.     

Unconventional interactions between water and heterocyclic nitrogens in protein structures

We report an unusual interaction in which a water molecule approaches the heterocyclic nitrogen of tryptophan and histidine along an axis that is roughly perpendicular to the aromatic plane of the side chain. The interaction is distinct from the well-known conventional aromatic hydrogen-bond, and it occurs at roughly the same frequency in protein structures. Calculations indicate that the water-indole interaction is favorable energetically, and we find several cases in which such contacts are conserved among structural orthologs. The indole-water interaction links side chains and peptide backbone in turn regions, connects the side chains in beta-sheets, and bridges secondary elements from different domains. We suggest that the water-indole interaction can be indirectly responsible for the quenching of tryptophan fluorescence that is observed in the folding of homeodomains and, possibly, many other proteins. We also observe a similar interaction between water and the imidazole nitrogens of the histidine side chain. Taken together, these observations suggest that the unconventional water-indole and water-imidazole interactions provide a small but favorable contribution to protein structures.     

Enzyme-mediated amperometric biosensors prepared with the Layer-by-Layer (LbL) adsorption technique

Glucose oxidase (GOD) has been immobilized in Layer-by-Layer (LbL) films, adsorbed alternately with poly(allylamine) hydrochloride (PAH) layers, onto an ITO substrate modified with a Prussian Blue (PB) layer. The ITO/PB/GOD-PAH heterostructures were tested in amperometric glucose biosensors, with a high sensitivity of 16 μA mmol⁻¹ l cm^-2 and a limit of detection of 0.20 mmol l⁻¹ being achieved. This high sensitivity is attributed to the ultrathin nature of the film in addition to the low operating potentials that could be used due to the efficient catalysis of H₂O₂ produced in the enzymatic reaction in the presence of Prussian Blue. The biosensors are highly selective to glucose, as demonstrated by the lack of interference from possible interferents such as ascorbic and uric acids and acetominophen. The stability of the biosensors was checked by observing an almost constant sensitivity for a period of approximately 20 days, thus indicating a stable adsorption of GOD.     

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

Tumor-induced endothelial cell activation: role of vascular endothelial growth factor

Proangiogenic, proliferative effects of tumors have been extensively characterized in subconfluent endothelial cells (EC), but results in confluent, contact-inhibited EC are critically lacking. The present study examined the effect of tumor-conditioned medium (CM) of the malignant osteoblastic cell line MG63 on monolayer, quiescent bovine aorta EC. MG63-CM and MG63-CM + CoCl₂ significantly increased EC survival in serum-starved conditions, without inducing EC proliferation. Furthermore, MG63-CM and MG63-CM + CoCl₂, both containing high amounts of vascular endothelial growth factor (VEGF), induced relevant phenotypic changes in EC (all P < 0.01) involving increase of nucleoli/chromatin condensations, nucleus-to-cytosol ratio, capillary-like vacuolated structures, vessel-like acellular areas, migration through Matrigel, growth advantage in reseeding, and factor VIII content. All these actions were significantly inhibited by VEGF and VEGF receptor (VEGFR2) blockade. Of particular importance, a set of similar effects were detected in a human microvascular endothelial cell line (HMEC). With regard to gene expression, incubation with MG63-CM abolished endogenous VEGF mRNA and protein but induced a clear-cut increase in VEGFR2 mRNA expression in EC. In terms of mechanism, MG63-CM activates protein kinase B (PKB)/Akt, p44/p42-mitogen-activated protein kinase (MAPK)-mediated pathways, as suggested by both inhibition and phosphorylation experiments. In conclusion, tumor cells activate confluent, quiescent EC, promoting survival, phenotypic, and gene expression changes. Of importance, VEGF antagonism converts MG63-CM from protective to EC-damaging effects. vascular endothelial growth factor receptor 2; MG63-conditioned medium     

Reduction of Glutamate Uptake into Cerebral Cortex of Developing Rats by the Branched-Chain Alpha-Keto Acids Accumulating in Maple Syrup Urine Disease

In the current study we investigated the effect of the branched-chain alpha-keto acids (BCKA) co-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV), and alpha-ketoisovaleric (KIV) acids, which accumulate in maple syrup urine disease (MSUD), on the in vitro uptake of [3H]glutamate by cerebral cortical slices from rats aged 9, 21, and 60 days of life. We initially observed that glutamate uptake into cerebral cortex of 9- and 21-day-old rats was significantly higher, as compared to that of 60-day-old rats. Furthermore, KIC inhibited this uptake by tissue slices at all ages studied, whereas KMV and KIV produced the same effect only in cortical slices of 21- and 60-day-old rats. Kinetic assays showed that KIC significantly inhibited glutamate uptake in the presence of high glutamate concentrations (50 microM and greater). We also verified that the reduction of glutamate uptake was not due to cellular death, as evidenced by tetrazolium salt and lactate dehydrogenase viability tests of cortical slices in the presence of the BCKA. It is therefore presumed that the reduced glutamate uptake caused by the BCKA accumulating in MSUD may lead to higher extracellular glutamate levels and potentially to excitotoxicity, which may contribute to the neurological dysfunction of the affected individuals.     

Genetic diversity (RAPD-PCR) of lactobacilli isolated from "Almagro" eggplant fermentations from two seasons

Genetic diversity of 323 strains of lactobacilli isolated from an Almagro eggplant manufacturing plant was analyzed by using random amplified polymorphic DNA (RAPD). Thirty-four distinct RAPD patterns were obtained with 95% of isolates grouped into 18 main clusters. Genetic diversity was higher in brines from season II, Lactobacillus plantarum and Lactobacillus fermentum/cellobiosus being the species with the greatest number of genotypes. A single L. fermentum/cellobiosus genotype comprised isolates from both seasons and could be considered endemic to that factory.     

Nitric oxide and iron: effect of iron overload on nitric oxide production in endotoxemia

The amount of iron within the cell is carefully regulated in order to provide an adequate level of micronutrient while preventing its accumulation and toxicity. Iron excess is believed to generate oxidative stress, understood as an increase in the steady-state concentration of oxygen radical intermediates. Nitric oxide (NO) is an inorganic free-radical gaseous molecule which has been shown over the last decade to play an unprecedented variety of roles in biological systems. The effect of nitrogen reactive species may explain the iron sequestration pattern that characterizes macrophages under inflammatory conditions. From a patho-physiological viewpoint, further studies are required to assess the usefulness of this mechanism to minimize formation and release of free radicals in diseased tissues. However, contrary to the deleterious effects of the reactive nitrogen oxide species formed from either NO/O(2) and NO/O(2)(-), it has been pointed out that NO shows antioxidant properties. A number of studies have described the complex relationships between iron and NO, but controversy remains as to the influence and significance of iron on inflammatory NO production. To explore the initial steps of the effects triggered by LPS administration in the presence of excess iron, male Wistar rats were treated with: lipopolysaccharide from Escherichia coli (serotype 0127:B8) (LPS); iron-dextran; or iron-dextran plus LPS and liver samples were taken after 6 h. EPR spectra of NO-Hb in the venous blood were determined at 77 K. Iron-dextran administered to rats intraperitoneally resulted predominantly in iron uptake by the liver Kupffer cells and led to an increased NO level in blood in the presence of LPS. Further studies will be required to assess the complex role of the Kupffer cells on iNOS induction and NO production.