Coordination of magnesium with adenosine 5′-diphosphate and triphosphate

³¹P NMR chemical shifts of salts of adenosine 5′-triphosphate and diphosphate: ATPH^2?₂2(Me₄N⁺) · H₂O, ATPH^2?₂2 Na⁺ · 3.5 H₂O, ATPH^2?₂Mg²⁺ · 4 H₂O, ATPH^2?₂Ca²⁺ · 2 H₂O, ADPH^2?2(Me₄N⁺) · H₂O and ADPH^2?Mg²⁺ · 4 H₂O have been measured in 0.02 M ²H₂O solutions at 145.7 MHz (22° C) at constant p²H values (8.20 and 6.20). The results are compared with those obtained from salts of adenosine 5′-monophosphate and other simpler phosphomonoesters, e.g. AMP^2?2(Me₄N⁺), AMP^2?Mg²⁺, AMPH^?Me₄N⁺ and (AMPH^?)₂Mg²⁺. It is concluded that the effects exerted by Mg²⁺ and Ca²⁺ on the ³¹P NMR shifts of dipoly- and tripolyphosphates relative to monovalent cations are due mainly to changes in conformation of the polyphosphate chain rather than to purely electronic factors associated with the binding of divalent cations to the phospho-oxyanions. The data are consistent with the existence of the following complexes at p²H 8.20: (MgP_αP_β)ADP^? and (MgP_αP_γ)ATP^2?af (MgP_αP_β)ATP^2?af (MgP_βP_γ)ATP^2? with the latter equilibrium relatively fast in the NMR time scale. Monoprotonation of the terminal phosphate appears to weaken the Mg²⁺-polyphosphate binding, particularly at P_β of MgADPH and at P_β and P_γ of MgATPH^?. The Mg²⁺-polyphosphate binding weakens further at p²H 3.70, i.e. in MgATPH₂. Possible implications of the results in the mechanism of actomyosin Mg²⁺-ATPase in muscle contraction are discussed.     

Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse

Background  

The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI).     

Nitrogen fluxes from treefrogs to tank epiphytic bromeliads: an isotopic and physiological approach

Diverse invertebrate and vertebrate species live in association with plants of the large Neotropical family Bromeliaceae. Although previous studies have assumed that debris of associated organisms improves plant nutrition, so far little evidence supports this assumption. In this study we used isotopic (¹⁵N) and physiological methods to investigate if the treefrog Scinax hayii, which uses the tank epiphytic bromeliad Vriesea bituminosa as a diurnal shelter, contributes to host plant nutrition. In the field, bromeliads with frogs had higher stable N isotopic composition (δ¹⁵N) values than those without frogs. Similar results were obtained from a controlled greenhouse experiment. Linear mixing models showed that frog feces and dead termites used to simulate insects that eventually fall inside the bromeliad tank contributed, respectively, 27.7% (±0.07 SE) and 49.6% (±0.50 SE) of the total N of V. bituminosa. Net photosynthetic rate was higher in plants that received feces and termites than in controls; however, this effect was only detected in the rainy, but not in the dry season. These results demonstrate for the first time that vertebrates contribute to bromeliad nutrition, and that this benefit is seasonally restricted. Since amphibian–bromeliad associations occur in diverse habitats in South and Central America, this mechanism for deriving nutrients may be important in bromeliad systems throughout the Neotropics.     

Fine mapping of a seizure susceptibility locus on mouse Chromosome 1: nomination of Kcnj10 as a causative gene

Previous quantitative trait loci (QTL) mapping studies document that the distal region of mouse Chromosome (Chr) 1 contains a gene(s) that is in large part responsible for the difference in seizure susceptibility between C57BL/6 (B6) (relatively seizure-resistant) and DBA/2 (D2) (relatively seizure-sensitive) mice. We now confirm this seizure-related QTL (Szs1) using reciprocal, interval-specific congenic strains and map it to a 6.6-Mb segment between Pbx1 and D1Mit150. Haplotype conservation between strains within this segment suggests that Szs1 may be localized more precisely to a 4.1-Mb critical interval between Fcgr3 and D1Mit150. We compared the coding region sequences of candidate genes between B6 and D2 mice using RT-PCR, amplification from genomic DNA, and database searching and discovered 12 brain-expressed genes with SNPs that predict a protein amino acid variation. Of these, the most compelling seizure susceptibility candidate is Kcnj10. A survey of the Kcnj10 SNP among other inbred mouse strains revealed a significant effect on seizure sensitivity such that most strains possessing a haplotype containing the B6 variant of Kcnj10 have higher seizure thresholds than those strains possessing the D2 variant. The unique role of inward-rectifying potassium ion channels in membrane physiology coupled with previous strong association between ion channel gene mutations and seizure phenotypes puts even greater focus on Kcnj10 in the present model. In summary, we confirmed a seizure-related QTL of large effect on mouse Chr 1 and mapped it to a finely delimited region. The critical interval contains several candidate genes, one of which, Kcnj10, exhibits a potentially important polymorphism with regard to fundamental aspects of seizure susceptibility.     

Chromosome Banding and 18S rDNA in situ Hybridization Analysis of Seven Species of the Family Achiridae (Teleostei: Pleuronectiformes)

Achiridae is an important family of the order Pleuronectiformes widely distributed in North, Central, and South America with freshwater and marine species. In the present study cytogenetic analyses comprising conventional and molecular techniques were carried out in seven species of this family. The following diploid numbers (2n) and fundamental numbers (FN) were obtained: Achirus declivis 2n = 34, FN = 52; Achirus lineatus 2n = 40, FN = 66; Catathyridium jenynsi 2n = 40 and FN = 50; Gymnachirus nudus 2n = 36 and FN = 50; Hypoclinemus mentalis 2n = 38 and FN = 54; Trinectes paulistanus 2n = 42 and FN = 52; and Trinectes sp. 2n = 38 and FN = 54. All species presented a single nucleolar organizer region (NOR) bearing chromosome pair and C-band positive segments mainly distributed at the pericentromeric position. The wide variation observed in chromosome number and FN suggests the occurrence of larger chromosome rearrangements in the family Achiridae if compared with other families of the same order.     

Sensitive liquid chromatography-mass spectrometry assay for quantitation of docetaxel and paclitaxel in human plasma

We have developed a high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS) method for quantifying docetaxel and paclitaxel in human plasma. The assay fulfills the need for defining the lower plasma concentrations of these antineoplastic agents that result from a number of changes in how these agents are used clinically. The assay uses paclitaxel as the internal standard for docetaxel, and vice versa; solid-phase extraction; a Phenomenex Hypersil ODS (5 micrometer, 100x2 mm) reversed-phase analytical column; an isocratic mobile phase of 0.1% formic acid in methanol-water (70:30, v/v); and mass spectrometric detection using electrospray positive mode electron ionization. The assay has a lower limit of quantitation (LLOQ) of 0.3 nM and is linear between 0.3 nM and 1 microM for docetaxel. For paclitaxel, the LLOQ was 1 nM, and the assay is linear between 1 nM and 1 microM. We demonstrated the suitability of this assay for docetaxel by using it to quantify the docetaxel concentrations in plasma of a patient given 40 mg/m(2) of docetaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. In a similar manner, the suitability of the assay for paclitaxel was demonstrated by using it to quantify the concentrations of paclitaxel in the plasma of a patient given 15 mg/m(2) of paclitaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. The LC-MS assay, which proved superior because of its greater sensitivity and relatively short (7 min) run time, should be an important tool for future pharmacokinetic analyses of docetaxel and paclitaxel.     

Helianthus tuberosus lectin reveals a widespread scaffold for mannose-binding lectins

BACKGROUND: Heltuba, a tuber lectin from the Jerusalem artichoke Helianthus tuberosus, belongs to the mannose-binding subgroup of the family of jacalin-related plant lectins. Heltuba is highly specific for the disaccharides Man alpha 1-3Man or Man alpha 1-2Man, two carbohydrates that are particularly abundant in the glycoconjugates exposed on the surface of viruses, bacteria and fungi, and on the epithelial cells along the gastrointestinal tract of lower animals. Heltuba is therefore a good candidate as a defense protein against plant pathogens or predators. RESULTS: The 2.0 A resolution structure of Heltuba exhibits a threefold symmetric beta-prism fold made up of three four-stranded beta sheets. The crystal structures of Heltuba in complex with Man alpha 1-3Man and Man alpha 1-2Man, solved at 2.35 A and 2.45 A resolution respectively, reveal the carbohydrate-binding site and the residues required for the specificity towards alpha 1-3 or alpha 1-2 mannose linkages. In addition, the crystal packing reveals a remarkable, donut-shaped, octahedral assembly of subunits with the mannose moieties at the periphery, suggesting possible cross-linking interactions with branched oligomannosides. CONCLUSIONS: The structure of Heltuba, which is the prototype for an extended family of mannose-binding agglutinins, shares the carbohydrate-binding site and beta-prism topology of its galactose-binding counterparts jacalin and Maclura pomifera lectin. However, the beta-prism elements recruited to form the octameric interface of Heltuba, and the strategy used to forge the mannose-binding site, are unique and markedly dissimilar to those described for jacalin. The present structure highlights a hitherto unrecognized adaptability of the beta-prism building block in the evolution of plant proteins.     

The effect of propranolol on cAMP concentration in the rat preoptic region during the wake-sleep cycle

In control conditions preoptic cAMP concentration during wakefulness was significantly higher than during synchronized sleep. No differences in nucleotide concentration were observed in the cerebral cortex. Propranolol decreases brain cAMP concentration. This change was associated with the suppression of the difference observed between wakefulness and synchronized sleep in the preoptic region.     

Comparative Analysis of a Mitochondrial DNA Control Region Fragment Amplified from Three Adriatic Flatfish Species and Molecular Phylogenesis of Pleuronectiformes

The 5′-end of the mitochondrial control region of three Pleuronectiformes from the Adriatic Sea, Platichthys flesus italicus (Adriatic flounder), Solea vulgaris (common sole), and Solea kleini (Klein's sole), was sequenced and compared with that of six other flatfish species from the families Pleuronectidae and Bothidae. The sequence structures of all flatfishes appear very similar and consist of alternate short segments with low, medium, and high rates of nucleotide substitution. Four conserved 19-bp repeats occur at the beginning of the European and Adriatic flounder sequences. The common occurrence of tandem arrays in fish control regions could be related to a stable secondary structure. Molecular phylogenetic relationships among Pleuronectiformes agree well with previous morphologic data at all taxonomic levels. Molecular analyses could therefore contribute to resolving phylogenetic and taxonomic debates within the Pleuronectiformes. Received December 1, 1997; accepted June 30, 1998.     

Feedback control of the limbs position during voluntary rhythmic oscillation

The mechanisms that control the limbs position during rhythmic voluntary oscillations were investigated in ten subjects, who were asked to synchronise the lower peak of their hand or foot rhythmic oscillations to a metronome beat. The efficacy of the “position control” was estimated by measuring the degree of synchronisation between the metronome signal and the requested limb position and how it was affected by changing both the oscillation frequency (between 0.4 and 3.0 Hz) and the limbs inertial properties. With the limbs unloaded, the lower peak of both the hand and foot oscillations lagged the metronome beat of a slight amount that remained constant over the whole frequency range (mean phase delay −13.2° for the hand and −4.7° for the foot). The constancy was obtained by phase-advancing, at each frequency increment, the electromyogram (EMG) activation with respect of the clock beat of the amount necessary to compensate for the simultaneous increase of the lag between the EMG and the movement, produced by the limb mechanical impedance. After loading of either limb, the increase of the oscillation frequency induced larger EMG-movement delays and the anticipatory compensation became insufficient, so that the movement progressively phase-lagged the clock beat. The above results have been accurately simulated by a neural network connected to a pendulum model that shared the same mechanical properties of the moving limb. The network compares a central command (the intended position) to the actual position of the effector and acts as a closed-loop proportional, integrative and derivative controller. It is proposed that the synchronisation of rhythmic oscillations of either the hand or the foot is sustained by a feed-back control that conforms the position of each limb to that encoded in the central voluntary command.