Construction and characterization of centric circular and acentric linear chromosomes in fission yeast

Summary Gene disruption and gap repair of chromosomal DNA have been frequently employed techniques in yeast genetics. To extend the possibility of using these gene manipulations for larger genomic regions, we have examined the maximal sizes of chromosomal DNA disrupted or repaired in vivo. Here we report a simple, potentially general, method for selectively deleting a 150 kb region, or gap-filling a 100 kb region, in the fission yeast genome. This enables the generation of acentric linear chromosomes by deletion, or the cloning of large functional centromeric DNAs into circular minichromosomes by gap-filling. The fidelity of the resulting gap-filling is high, judging from partial-digestion mapping of gap-repaired DNAs. By analysing a series of such circular minichromosomes, we conclude that only a part of the repetitive centromeric region, including the central domain, is essential for mitotic and meiotic chromosome segregation. Acentric linear chromosomes, although unstable, could be maintained, indicating that it may be possible to construct an acentric vector for large DNA fragments in this organism.     

Influence of the parameters of several pulse sequences on the image contrast: comparison between the two major contrast media

We compared the influences of the parameters of several pulse sequences using two major commercially available gadolinium (Gd) contrast media for MR imaging. The phantom of Gd solutions of various concentration (0.1 - 10mmol/L) was prepared, and was scanned with a 1.5T clinical MR unit, using a spin-echo T1-weighted sequence, 2DFLASH, 3DFLASH, and 3DVIBE. The signal intensity was measured and the contrast enhancement ratio (CER) was calculated and plotted as a function of Gd concentration. The results were compared between the pulse sequences, and between the contrast media as well. Both 3DFLASH and 3DVIBE showed higher CER than other two sequences, showing similar CER curve configuration. There was no significant difference both in CER value and CER curve configuration between the two contrast media for each pulse sequence.     

Masked excitatory action of noradrenaline on rat islet β-cells via activation of phospholipase C

The effect of noradrenaline (NE) on rat islet -cells was examined. NE reduced insulin secretion from rat islets exposed to extracellular solutions containing glucose at 5.5 or 16.6 mM. In islets treated with pertussis toxin (PTX), however, NE increased insulin secretion. The NE-induced augmentation of insulin secretion was inhibited by prazosin. In intact islets, NE increased phospholipase C (PLC) activity, an effect that was prevented by treatment of islets with U-73122. NE elevated intracellular [Ca²⁺] ([Ca²⁺]_i) in isolated -cells independently of PTX. Although this NE effect was inhibited by prazosin, phenylephrine did not mimic it. The [Ca²⁺]_i response to NE was also prevented by the treatment of cells with U-73122. NE produced depolarization of -cells followed by nifedipine-sensitive action potentials. NE reduced the whole-cell membrane currents through ATP-sensitive K⁺ channels (K_ATP), responsible for the depolarization. This NE effect was prevented by treatment of -cells with U-73122 or BAPTA/AM. Although at least some of our results imply the presence of ₁-adrenoceptors, -cells were not stained by a polyclonal IgG antibody recognizing all adrenergic ₁-receptor subtypes so far identified. These results suggest that an interaction of NE with an unknown type of receptor activates rat islet -cells via a PLC-dependent signal pathway. This effect is, however, masked by the inhibitory action via a PTX-sensitive pathway also activated by NE.     

The role of IL-17 in systemic lupus erythematosus and its potential as a therapeutic target

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibodies production and immune complex deposition with systemic clinical manifestations. Interleukin (IL)-17-producing cells play a crucial role in disease pathogenesis and represent an attractive therapeutic target.

Areas covered: This review provides an update on the possibility of targeting IL-17 in SLE. The rational for this approach as well as currently available and future targets are discussed.

Expert opinion: Although human expression studies and animal models indicate that IL-17 blocking may be a promising therapeutic strategy for SLE, direct evidence for IL-17 inhibition in SLE patients is unavailable. Biologic therapies and small-molecule drugs that target IL-17 production are required for the achievement of a favorable clinical effect in SLE patients.     

Suppression of cell attachment and protein adsorption onto amphiphilic polylactide-grafted dextran films

To develop novel biodegradable biomedical materials, polylactide-grafted dextrans (Dex-g-PLA)s having various lengths, numbers of graft chains and sugar units were synthesized using the trimethylsilyl (TMS) protection method. To explore the possibility of using Dex-g-PLA as a biomedical soft-material, the contact angle, cell attachment and protein adsorption properties of the films prepared from these biodegradable and amphiphilic graft co-polymers were investigated. The poly-L-lactide (PLLA) film did not absorb water at all because of its high hydrophobicity, while the graft co-polymer films started immediately to swell after immersion in PBS. The percentage of water absorption at equilibrium increased with increasing sugar unit content. The receding contact angle of the Dex-g-PLA films against water was smaller than that of the PLLA film. The receding contact angle of Dex-g-PLA films against water decreased with increasing the sugar unit content. The top surface of the Dex-g-PLA film was suggested to be covered with hydrophilic Dex segments by means of annealing in water and to afford the wettable surface. Such a wettable surface led to the suppression of cell attachment and protein adsorption onto the film.     

Human neutrophils express messenger RNA of vitamin D receptor and respond to 1alpha,25-dihydroxyvitamin D3

1Alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to modulate the production of various cytokines or the expression of certain differentiation markers in human T cells or monocytes. Its effects on neutrophils, however, are poorly understood. In this paper, we show several lines of evidence indicating that neutrophils express functional vitamin D receptors (VDR). Sort-purified neutrophils from human peripheral blood expressed VDR mRNA at a level comparable to that of monocytes. As reported to occur in monocytes, protein expression of CD14 on the cell surface of neutrophils was augmented when the cells were incubated with 1,25(OH)2D3. To investigate the physiological roles for VDR in neutrophils, we investigated possible modulating effects of 1,25(OH)2D3 on the expression of several genes in lipopolysaccharide-stimulated neutrophils by using differential display analysis. Of the genes we identified, trappin-2/elafin/SKALP, which was originally reported to be an inhibitor of elastase, was induced in neutrophils by lipopolysaccharide, but was suppressed significantly in the presence of 1,25(OH)2D3. Under the same conditions, interleukin-1beta expression was also inhibited. These findings suggest that 1,25(OH)2D3 has a potential to affect the inflammatory process by modulating the expression of neutrophil genes.     

Effects of reduced frequency breathing on arterial hypoxemia during exercise

Summary It is uncertain that exercise with reduced frequency breathing (RFB) results in arterial hypoxemia. This study was designed to investigate whether RFB during exercise creates a true hypoxic condition in arterial blood by examining arterial oxygen saturation (SaO₂) directly. Six subjects performed ten 30 s periods of exercise on a Monark bicycle ergometer at a work rate of 210 W alternating with 30 s rest intervals. The breath was controlled to use 1 s each for inspiration and expiration, and two trials with different breathing patterns were used; a continuous breathing (CB) trial and an RFB trial consisting of four seconds of breath-holding at functional residual capacity (FRC). Alveolar oxygen pressure during exercise showed a slight but significant (p<0.05) reduction with RFB as compared to CB. However, a marked increase in alveolar-arterial pressure difference for oxygen (A-aDO₂) (p<0.05) with RFB over CB resulted in a marked (p<0.05) reduction in arterial oxygen pressure. Consequently, SaO₂ fell as low as 88.8% on average. Additional examination of RFB with breath-holding at total lung capacity showed no increases in A-aDO₂ in spite of the same amount of hypoventilation as compared with that at FRC. These results indicate that RFB during exercise can result in arterial hypoxemia if RFB is performed with breath-holding at FRC, this mechanism being closely related to the mechanical responses due to lung volume restriction.     

Suppressive effect of leflunomide metabolite (A77 1726) on metalloproteinase production in IL-1beta stimulated rheumatoid synovial fibroblasts

Leflunomide, an isoxazol derivative structurally unrelated to other immunomodulatory drugs, has proven to be efficacious in the treatment of rheumatoid arthritis (RA). This study was conducted to elucidate the mechanism by which leflunomide mediated antirheumatic effects. We investigated the effects of A77 1726, leflunomide's active metabolite, on mitogen-activated protein kinase (MAPK) activation in IL-1beta-stimulated rheumatoid synovial fibroblasts. The effects of A77 1726 on the secretion of matrix metalloproteinases (MMPs) from rheumatoid synovial fibroblasts were also examined. A77 1726 partially suppressed IL-1beta-induced ERK1/2 and p38 kinase activation. In contrast, A77 1726 efficiently suppressed IL-1beta-stimulated JNK1/2 kinase activation. Although no suppressive effect was demonstrated on MMP-2, A77 1726 markedly inhibited MMP-1, 3, and 13 secretions from IL-1beta-stimulated rheumatoid synovial fibroblasts. Tissue inhibitor of metalloproteinases-1 (TIMP-1) was constitutively produced from rheumatoid synovial fibroblasts and the suppressive effects of A77 1726 on TIMP-1 production were minimal. Our results suggest that the suppression of the MAPK signalling pathway and MMP synthesis in rheumatoid synovial fibroblasts is a possible mechanism for the inhibitory activity of leflunomide against rheumatoid arthritis.     

Characterization of Chlamydia pneumoniae species-specific proteins immunodominant in humans.

Proteins of Chlamydia pneumoniae immunodominant in humans were characterized with the sera of 13 patients who were not likely to have been exposed to C. trachomatis or C. psittaci. The serological responses among these patients were similar on a qualitative basis, but some differences were found quantitatively. However, the serological responses of the patients who were infected with C. pneumoniae differed markedly from those of two patients who were infected with C. trachomatis and two who were infected with C. psittaci and those of mice that were transtracheally infected with C. pneumoniae. Among proteins immunodominant in the patients who were infected with C. pneumoniae, a 40-kDa major outer membrane protein was genus specific and 53-, 46-, and 43-kDa proteins were species specific in their reactions with the majority of the human sera used. A few sera reacted strongly with a 73-kDa protein genus specifically. Some proteins with weak immunogenicity exhibited species specificity. An antigenic analysis with human sera and murine monoclonal antibodies against the 53-kDa protein showed that hte antigenicities were strictly conserved among the seven strains of C. pneumoniae tested. The genus-specific 73-kDa protein was solubilized with octylglucoside. All of the species-specific immunodominant proteins were solubilized with sodium dodecyl sulfate, but the genus-specific major outer membrane protein was not. These results suggest that a serological diagnosis of C. pneumoniae infection could be achieved species specifically by comparison of the serum responses to sodium dodecyl sulfate- and octylglucoside-soluble fractions.     

A novel translocation involving chromosomes 2, 9, 14, and 22 in chronic myeloid leukemia

A 46-year-old man with chronic myelogenous leukemia was found to have a new complex translocation. In chronic phase, all of the bone marrow cells had a rearrangement of a t(2;9;14;22) (p21;q34;q32;q11). Southern blot analysis of leukocyte DNA revealed rearrangement of the breakpoint cluster region (bcr) within the 5.8-Kb bcr. The patient eventually died in blast crisis 28 months later. The cytogenetic findings of bone marrow cells showed a 46,XY,t(2;9;14;22)(p21;q34;q32;qll),add(lp),del(3q) karyotype in blast crisis.