Comparative FISH mapping on Z chromosomes of chicken and Japanese quail

Using direct R-banding fluorescence in situ hybridization, we assigned five functional genes-growth hormone receptor (GHR), prolactin receptor (PRLR), spleen tyrosine kinase (SYK), aldolase B (ALDOB), and muscle skeletal receptor tyrosine kinase (MUSK)-to the chicken Z chromosome. SYK and MUSK were newly localized to the chicken Z chromosome in this study. GHR and PRLR were situated close to each other on the short arm of the chicken Z chromosome, as are their counterparts on human chromosome 5. SYK, MUSK, and ALDOB, which have been mapped to human chromosome 9, were localized to the long arm of the chicken Z chromosome. Thus, the present results indicate the presence of conserved synteny between the chicken Z chromosome and human chromosomes 5 and 9. Using the same method, four of the genes (GHR, PRLR, ALDOB, and MUSK) were assigned to the Japanese quail Z chromosome. The locations of these four Z-linked genes were conserved between chicken and Japanese quail. The results support the notion that the avian Z chromosome and the mammalian X chromosome did not evolve from a common ancestral linkage group.     

Electrochemical sensing of the behaviour of oligonucleotide lipoplexes at charged interfaces

Complexes between short oligodeoxynucleotides (ODN) with a variable dG(x)dC(y) base composition and liposomes composed of the cationic lipid DOTAP (ODN lipoplexes) were studied by differential pulse voltammetry at a glassy carbon electrode. Since lipoplexes are spontaneously formed by electrostatic interactions, the objective of the voltammetric study was to investigate their behaviour at the electrode surface/solution interface. It was verified that the peak current in the voltammograms for ODN lipoplexes was due to guanosine oxidation and that it was influenced both by the applied adsorption potential and the lipoplex (+/-) charge ratio used. It was found that for low ODN lipoplexes (+/-) charge ratios the peak current obtained was enhanced when compared to that registered with free ODN for the same concentration. This allowed a higher sensitivity in the determination of ODN by differential pulse voltammetry and a limit of detection of 5.5 ng/mL was achieved. A model that explains the organisation of ODN lipoplexes at the electrode surface/solution interface is proposed. The electrochemical results presented account for a better physicochemical characterisation of lipoplexes at charged interfaces, which can be important for the understanding and development of gene therapy vectors based on ODN lipoplexes.     

Bed rest attenuates sympathetic and pressor responses to isometric exercise in antigravity leg muscles in humans

Although spaceflight and bed rest are known to cause muscular atrophy in the antigravity muscles of the legs, the changes in sympathetic and cardiovascular responses to exercises using the atrophied muscles remain unknown. We hypothesized that bed rest would augment sympathetic responses to isometric exercise using antigravity leg muscles in humans. Ten healthy male volunteers were subjected to 14-day 6 degrees head-down bed rest. Before and after bed rest, they performed isometric exercises using leg (plantar flexion) and forearm (handgrip) muscles, followed by 2-min postexercise muscle ischemia (PEMI) that continues to stimulate the muscle metaboreflex. These exercises were sustained to fatigue. We measured muscle sympathetic nerve activity (MSNA) in the contralateral resting leg by microneurography. In both pre- and post-bed-rest exercise tests, exercise intensities were set at 30 and 70% of the maximum voluntary force measured before bed rest. Bed rest attenuated the increase in MSNA in response to fatiguing plantar flexion by approximately 70% at both exercise intensities (both P < 0.05 vs. before bed rest) and reduced the maximal voluntary force of plantar flexion by 15%. In contrast, bed rest did not alter the increase in MSNA response to fatiguing handgrip and had no effects on the maximal voluntary force of handgrip. Although PEMI sustained MSNA activation before bed rest in all trials, bed rest entirely eliminated the PEMI-induced increase in MSNA in leg exercises but partially attenuated it in forearm exercises. These results do not support our hypothesis but indicate that bed rest causes a reduction in isometric exercise-induced sympathetic activation in (probably atrophied) antigravity leg muscles.     

Identification of QTLs controlling tissue-culture traits in barley ( Hordeum vulgare L.)

Quantitative trait loci (QTLs) controlling callus growth (CG), subsequent shoot differentiation ratio (SD) and green shoot ratio (GS) in immature embryo culture were identified in barley. A base map was developed from 99 recombinant inbred lines (RILs) of 'Azumamugi' 2 'Kanto Nakate Gold'. The tissue-culture traits were evaluated at the F₇ and F₁₀ generations of the RILs. The RILs showed wide and continuous variations in each of the three tissue-culture traits. Three QTLs for CG, three QTLs for SD and two QTLs for GS were detected by using composite interval mapping. A QTL for SD on chromosome 3H had a large effect, and 'Kanto Nakate Gold', which has a high differentiation ability, contributed to this QTL. The location of this QTL is identical to, or very close to, the uzu locus. We discuss the relationships between tissue-culture loci in 'Azumamugi' 2 'Kanto Nakate Gold' and those in other mapping populations.     

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.     

Differential electron flow around photosystem I by two C(4)-photosynthetic-cell-specific ferredoxins

In the C(4) plant maize (Zea mays L.), two ferredoxin isoproteins, Fd I and Fd II, are expressed specifically in mesophyll and bundle-sheath cells, respectively. cDNAs for these ferredoxins were introduced separately into the cyanobacterium Plectonema boryanum with a disrupted endogenous ferredoxin gene, yielding TM202 and KM2-9 strains expressing Fd I and Fd II. The growth of TM202 was retarded under high light (130 micromol/m(2)/s), whereas KM2-9 grew at a normal rate but exhibited a nitrogen-deficient phenotype. Measurement of photosynthetic O(2) evolution revealed that the reducing power was not efficiently partitioned into nitrogen assimilation in KM2-9. After starvation of the cells in darkness, the P700 oxidation level under far-red illumination increased significantly in TM202. However, it remained low in KM2-9, indicating an active cyclic electron flow. In accordance with this, the cellular ratio of ATP/ADP increased and that of NADPH/NADP(+) decreased in KM2-9 as compared with TM202. These results demonstrated that the two cell type-specific ferredoxins differentially modulate electron flow around photosystem I.     

Endogenous production of TGF-beta is essential for osteoclastogenesis induced by a combination of receptor activator of NF-kappa B ligand and macrophage-colony-stimulating factor

Differentiation of osteoclasts, the cells primarily responsible for bone resorption, is controlled by a variety of osteotropic hormones and cytokines. Of these factors, receptor activator of NF-kappaB (RANK) ligand (RANKL) has been recently cloned as an essential inducer of osteoclastogenesis in the presence of M-CSF. Here, we isolated a stroma-free population of monocyte/macrophage (M/Mphi)-like hemopoietic cells from mouse unfractionated bone cells that were capable of differentiating into mature osteoclasts by treatment with soluble RANKL (sRANKL) and M-CSF. However, the efficiency of osteoclast formation was low, suggesting the requirement for additional factors. The isolated M/Mphi-like hemopoietic cells expressed TGF-beta and type I and II receptors of TGF-beta. Therefore, we examined the effect of TGF-beta on osteoclastogenesis. TGF-beta with a combination of sRANKL and M-CSF promoted the differentiation of nearly all M/Mphi-like hemopoietic cells into cells of the osteoclast lineage. Neutralizing anti-TGF-beta Ab abrogated the osteoclast generation. These TGF-beta effects were also observed in cultures of unfractionated bone cells, and anti-TGF-beta blocked the stimulatory effect of 1, 25-dihydroxyvitamin D(3). Translocation of NF-kappaB into nuclei induced by sRANKL in TGF-beta-pretreated M/Mphi-like hemopoietic cells was greater than that in untreated cells, whereas TGF-beta did not up-regulate the expression of RANK, the receptor of RANKL. Our findings suggest that TGF-beta is an essential autocrine factor for osteoclastogenesis.     

Characterization of Japanese eel immunoglobulin M and its level in serum

Japanese eel immunoglobulin M (IgM) was purified from the sera of Anguilla japonica immunized with Edwardsiella tarda FPU 347 and characterized. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) under reducing and non-reducing conditions revealed that the eel IgM was a tetrameric protein with a molecular weight of 790 000; it contained an equimolar heavy chain and light chain with molecular weights of 72 000 and 25 000, respectively. While the N-terminal sequence of the heavy chain, VELTQPGSMVLKPGQSLTI, showed similarity to the variable regions of those of teleost fishes Igs, the N-terminal sequence of the light chain, DIVLTQSPAVQSVQLGDT, was similar to the variable regions of chondrostei and mammalian kappa chains. Lectin-binding assays showed that the binding of concanavalin A (Con A) to the Japanese eel IgM heavy chain was competitively inhibited by -mannose and could be abolished by α-mannosidase treatment indicating the presence on the heavy chain of oligosaccharides, whose terminal were a bound mannoses. The average IgM concentration in the sera of the healthy eels was 3.4 mg ml⁻¹; it amounted to 10.3% of the total serum protein.     

Heterogeneous distribution of plasma membrane glycoconjugates in pancreatic acinar cells

Flow-cytometric studies of lectin binding to individual acinar cells have been carried out in order to analyse the distribution of membrane glycoconjugates in cells from different areas of the pancreas: duodenal lobule (head) and splenic lobule (body and tail). The following fluoresceinated lectins were used: wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine and sialic acid, L-fucose and D-mannose, respectively. In both pancreatic areas, two cell populations (R1 and R2) were identified according to the forward scatter (size). On the basis of their glycoconjugate pattern, R1 cells displayed higher density of WGA and TP receptors than R2 cells throughout the pancreas. Although no difference in size was found between the cells from duodenal and splenic lobules, N-acetyl D-glucosamine and/or sialic acid and L-fucose residues were more abundant in plasma membrane cell glycoconjugates from the duodenal lobule. The results provide evidence for biochemical heterogeneity among individual pancreatic cells according to the distribution of plasma membrane glycoconjugates.     

Tec/Bmx non-receptor tyrosine kinases are involved in regulation of Rho and serum response factor by Galpha12/13.

A transient transfection system was used to identify regulators and effectors for Tec and Bmx, members of the Tec non-receptor tyrosine kinase family. We found that Tec and Bmx activate serum response factor (SRF), in synergy with constitutively active alpha subunits of the G12 family of GTP-binding proteins, in transiently transfected NIH 3T3 cells. The SRF activation is sensitive to C3, suggesting the involvement of Rho. The kinase and Tec homology (TH) domains of the kinases are required for SRF activation. In addition, kinase-deficient mutants of Bmx are able to inhibit Galpha13- and Galpha12-induced SRF activation, and to suppress thrombin-induced SRF activation in cells lacking Galphaq/11, where thrombin's effect is mediated by G12/13 proteins. Moreover, expression of Galpha12 and Galpha13 stimulates autophosphorylation and transphosphorylation activities of Tec. Thus, the evidence indicates that Tec kinases are involved in Galpha12/13-induced, Rho-mediated activation of SRF. Furthermore, Src, which was previously shown to activate kinase activities of Tec kinases, activates SRF predominantly in Rho-independent pathways in 3T3 cells, as shown by the fact that C3 did not block Src-mediated SRF activation. However, the Rho-dependent pathway becomes significant when Tec is overexpressed.