Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.     

Diarrhea-associated HIV-1 APIs potentiate muscarinic activation of Cl- secretion by T84 cells via prolongation of cytosolic Ca2+ signaling

Aspartyl protease inhibitors (APIs) effectively extend the length and quality of life in human immunodeficiency virus (HIV)-infected patients, but dose-limiting side effects such as lipodystrophy, insulin resistance, and diarrhea have limited their clinical utility. Here, we show that the API nelfinavir induces a secretory form of diarrhea in HIV-infected patients. In vitro studies demonstrate that nelfinavir potentiates muscarinic stimulation of Cl^- secretion by T84 human intestinal cell monolayers through amplification and prolongation of an apical membrane Ca²⁺-dependent Cl^- conductance. This stimulated ion secretion is associated with increased magnitude and duration of muscarinically induced intracellular Ca²⁺ transients via activation of a long-lived, store-operated Ca²⁺ entry pathway. The enhanced intracellular Ca²⁺ signal is associated with uncoupling of the Cl^- conductance from downregulatory intracellular mediators generated normally by muscarinic activation. These data show that APIs modulate Ca²⁺ signaling in secretory epithelial cells and identify a novel target for treatment of clinically important API side effects. nelfinavir; clotrimazole; barium     

A Highly Active ATP-Insensitive K+ Import Pathway in Plant Mitochondria

We describe here a regulated and highly active K+ uptake pathway in potato (Solanum tuberosum), tomato (Lycopersicon esculentum), and maize (Zea mays) mitochondria. K+ transport was not inhibited by ATP, NADH, or thiol reagents, which regulate ATP-sensitive K+ channels previously described in plant and mammalian mitochondria. However, K+ uptake was completely prevented by quinine, a broad spectrum K+ channel inhibitor. Increased K+ uptake in plants leads to mitochondrial swelling, respiratory stimulation, heat release, and the prevention of reactive oxygen species formation. This newly described ATP-insensitive K+ import pathway is potentially involved in metabolism regulation and prevention of oxidative stress.     

Effects of high static magnetic field exposure on different DNAs

The effects of magnetic fields produced by permanent magnets on different DNA sources were investigated in vivo and in vitro. Escherichia coli DNA, plasmid, and amplification products of different lengths were used as the magnetic field target. The in vivo assays did not reveal any DNA alterations following exposure, demonstrating the presence of cell dependent mechanisms, such as the repair system and the buffering action of the heat shock proteins DNA K/J (Hsp 70/40). The in vitro assays displayed interactions between the magnetic field and DNA, revealing principally that magnetic field exposure induces DNA alterations in terms of point mutations. We speculate that the magnetic field can perturb DNA stability interacting with DNA directly or potentiating the activity of oxidant radicals. This genotoxic effect of the magnetic field, however, is minimized in living organisms due to the presence of protective cellular responses.     

Enantioselective pharmacokinetics of lercanidipine in healthy volunteers

OBJECTIVE: The enantioselective kinetic disposition of lercanidipine, a dihydropyridine type of third-generation calcium antagonist, was investigated in six healthy male volunteers following a single 20 mg racemic oral dose. METHODS: Serial plasma samples were obtained from 0 to 24 h after drug administration. Lercanidipine enantiomers were analysed using a chiral LC-MS-MS method. RESULTS: The following differences (p < 0.05, Wilcoxon test) between (S) and (R) enantiomers were found (median): C(max) 2.071 ng mL(-1) versus 1.681 ng mL(-1); AUC(0-24)12.352 ng h mL(-1) versus 10.063 ng h mL(-1) and Cl/f 732.16 L h(-1) versus 1891.84 L h(-1). The AUC(0-infinity) values for (S)-LER were 1.21-fold higher than those for (R)-LER. CONCLUSION: The pharmacokinetics of LER was enantioselective in healthy volunteers following a single dose of 20 mg of the unlabeled racemic drug.     

1H magnetization transfer in hydrated gluten and flour: effects of wheat aging

The interaction of water with flour or gluten in hydrated samples was investigated by proton magnetization transfer measurements. Flour and gluten from both durum and bread wheat seeds, either unaged or artificially aged over different periods of time, were investigated. Measurements were performed at several radio frequency power levels and frequency offsets, and the data were quantitatively modeled by two interacting pools, a liquid (water) and a solid (macromolecules) one. A super-Lorentzian line shape well described the magnetization of the solid pool. Magnetization transfer was found to be more efficient for flour with respect to gluten samples, in agreement with their hydrophilic/hydrophobic behavior. The aging treatment of seeds resulted in a minor degree of interaction between macromolecules and water.     

Biomechanical analysis of pedalling for rehabilitation purposes: experimental results on two pathological subjects and comparison with non-pathological findings

In this paper the experimental results obtained by means of a prototype measuring device dedicated to the evaluation of the rehabilitation level of the lower limb are presented. The analysis of the experimental data collected on non-pathological subjects allows the identification of the characteristic meaning of the most significant parameters typical of healthy subjects. These data have been employed for a systematic comparison with the same parameters measured on two pathological subjects, in order to define quantitative indicators of the rehabilitation degree of the lower limbs and indicators of the "quality" of the movement.     

Ventilatory frequency indicates visual recognition of an allopatric predator in nai;ve Nile tilapia

Perceiving a possible predator may promote physiological changes to support prey ‘fight or flight’. In this case, an increase in ventilatory frequency (VF) may be expected, because this is a way to improve oxygen uptake for escape tasks. Therefore, changes in VF may be used as a behavioral tool to evaluate visual recognition of a predator threat. Thus, we tested the effects of predator visual exposure on VF in the fish Nile tilapia, Oreochromis niloticus. For this, we measured tilapia VF before and after the presentation of three stimuli: an aquarium with a harmless fish or a predator or water (control). Nile tilapia VF increased significantly in the group visually exposed to a predator compared with the other two, which were similar to each other. Hence, we conclude that Nile tilapia may recognize an allopatric predator; consequently VF is an effective tool to indicate visual recognition of predator threat in fish.     

Differential regulation of the release of tumor necrosis factor-alpha and of eicosanoids by mast cells in rat airways after antigen challenge

BACKGROUND: Rat trachea display a differential topographical distribution of connective tissue mast cells (CTMC) and mucosal mast cells (MMC) that may imply regional differences in the release of allergic mediators such as tumor necrosis factor-alpha (TNF-alpha) and eicosanoids. AIM: To evaluate the role of CTMC and MMC for release of TNF-alpha and eicosanoids after allergenic challenge in distinct segments of rat trachea. MATERIALS AND METHODS: Proximal trachea (PT) and distal trachea (DT) from ovalbumin (OVA)-sensitized rats, treated or not with compound 48/80 (48/80) or dexamethasone, were incubated in culture medium. After OVA challenge, aliquots were collected to study release of TNF-alpha and eicosanoids. RESULTS: Release of TNF-alpha by PT upon OVA challenge peaked at 90 min and decayed at 6 and 24 h. Release from DT peaked at 30-90 min and decayed 6 and 24 h later. When CTMC were depleted with 48/80, OVA challenge exacerbated the TNF-alpha release by PT at all time intervals, while DT exacerbated TNF-alpha levels 6 and 24 h later only. Dexamethasone reduced TNF-alpha production after 90 min of OVA challenge in PT and at 3 and 6h in DT. OVA challenge increased prostaglandin D2) in DT and leukotriene B4 in both segments but did not modify prostaglandin E2 and leukotriene C4 release. CONCLUSION: OVA challenge induces TNF-alpha release from MMC, which is negatively regulated by CTMC. The profile of TNF-alpha and eicosanoids depends on the time after OVA challenge and of the tracheal segment considered.