Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Involvement of aquaporin in thromboxane A2 receptor-mediated, G12/13/RhoA/NHE-sensitive cell swelling in 1321N1 human astrocytoma cells

The physiological role of the thromboxane A₂ (TXA₂) receptor expressed on glial cells remains unclear. We previously reported that 1321N1 human astrocytoma cells pretreated with dibutyryl cyclic AMP (dbcAMP) became swollen in response to U46619, a TXA₂ analogue. In the present study, we examined the detailed mechanisms of TXA₂ receptor-mediated cell swelling in 1321N1 cells. The cell swelling caused by U46619 was suppressed by expression of p115-RGS, an inhibitory peptide of Gα_12/13 pathway and C3 toxin, an inhibitory protein for RhoA. The swelling was also inhibited by treatment with Y27632, a Rho kinase inhibitor and 5-(ethyl-N-isopropyl)amiloride (EIPA), a Na⁺/H⁺-exchanger inhibitor. Furthermore, cell swelling was suppressed by the pretreatment with aquaporin inhibitors mercury chloride or phloretin in a concentration-dependent manner, suggesting that aquaporins are involved in U46619-induced 1321N1 cell swelling. In fact, U46619 caused [³H]H₂O influx into the cells, which was inhibited by p115-RGS, C3 toxin, EIPA, mercury chloride and phloretin. This is the first report that the TXA₂ receptor mediates water influx through aquaporins in astrocytoma cells via TXA₂ receptor-mediated activation of Gα_12/13, Rho A, Rho kinase and Na⁺/H⁺-exchanger.     

Environmental and genetic control of crassulacean acid metabolism in two crassulacean species and an F1 hybrid with differing biomass δ13C values

Abstract The growth, biomass δ¹³C values, and ability to accumulate titratable acidity at night were compared in eight environmental treatments for Cremnophila linguifolia, Sedum greggii, and their F₁ hybrid. In the phytotron, differences in treatment daylength, day/night temperature and water availability were all found to have effects on total plant dry weight, nocturnal accumulation of titratable acidity and biomass δ¹³C value of at least some of the genotypes. However, there were differences between the genotypes both in the magnitude and direction of response of the phenotypic properties to the treatment variables. The phytotron δ¹³C values ranged from -12.9 to -19.2‰ for C. linguifolia, from -22.2 to -33.4‰ for S. greggii, and from -19.2 to -24.9‰ for the hybrid. After with-holding water for 76 h both C. linguifolia and the hybrid had midday Ψ_leaf values of -0.23 MPa; however, S. greggii had a value of -1.05 MPa. In contrast to past observations of other species, the daily watered plants of C. linguifolia had less negative δ¹³C values than did the plants watered only weekly.     

Short hairpin RNAs against eotaxin or interleukin‐5 decrease airway eosinophilia and hyper‐responsiveness in a murine model of asthma

Background

Eosinophilia plays the major role in the pathogenesis of asthma and correlates with the up‐regulation of eotaxin, which, together with interleukin (IL)‐5, is important for differentiation, chemo‐attraction, degranulation, and survival of eosinophils in local tissue. In a previous study, we found that administration of lentivirus‐delivered short hairpin RNA (shRNA) to suppress the expression of IL‐5 inhibited airway inflammation. The present study aimed to investigate the role of eotaxin shRNA and the synergistic effect of eotaxin and IL‐5 shRNAs on airway inflammation in an ovalbumin (OVA)‐induced murine model of asthma.

Methods

Lentivirus‐delivered shRNAs were used to suppress the expression of eotaxin and/or IL‐5 in local tissue in an OVA‐induced murine asthma model.

Results

Intra‐tracheal administration of lentivirus containing eotaxin shRNA expressing cassette (eoSEC3.3) efficiently moderated the characteristics of asthma, including airway hyper‐responsiveness, cellular infiltration of lung tissues, and eotaxin and IL‐5 levels in bronchio‐alveolar lavage fluid. Administration of lentiviruses expressing IL‐5 or eotaxin shRNAs (IL5SEC4 + eoSEC3.3) also moderated the symptoms of asthma in a mouse model.

Conclusions

Local delivery of lentiviruses expressing IL‐5 and eotaxin shRNAs provides a potential tool in moderating airway inflammation and also has the potential for developing clinical therapy based on the application of shRNAs of chemokines and cytokines involved in T helper 2 cell inflammation and eosinophilia. Copyright © 2008 John Wiley & Sons, Ltd.     

An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis

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Allogibberic acid: An inhibitor of flowering in Lemna perpusilla

Allogibberic acid (I) has been identified as the compound responsible for the inhibition of flowering, increase in frond multiplication rate and decrease in frond size produced in Lemna perpusilla 6746 by autoclaved, unbuffered aqueous solutions of gibberellic acid (VII). 13-Deoxyallogibberic acid (IV), a product of autoclaving aq. GA₇ (VIII) solutions, also inhibits flowering in L. perpusilla and is about 10 times more active than allogibberic acid.     

In vivo antiplasmodial activity of 11(13)-dehydroivaxillin from Carpesium ceruum

The whole plants of Carpesium genus are used in traditional medicine as anti-pyretic, analgesic and vermifugic, including a topical application for sores and inflammation. A previous study on Carpesium genus suggested that the antiplasmodial activity against Plasmodium falciparum was due to the existence of 11(13)- dehydroivaxillin (DDV) from EtOAc extracts of C. ceruum (Compositae). Here, the antimalarial activity of DDV was evaluated against Plasmodium berghei in mice. The LD₅₀ of the compound was determined as 51.2 mg/kg, while doses of 124 mg/kg and above were found to be lethal to mice. DDV (2, 5, 10 mg/kg/day) exhibited a significant blood schizontocidal activity in 4-day early infection, repository evaluation and in an established infection with a significant mean survival time comparable to that of the standard drug, chloroquine, 5 mg/kg/day. DDV possesses a promising antiplasmodial activity, which can be exploited in malaria therapy.     

Development of membrane-potential responsiveness by myeloid leukemia cells during neutrophilic differentiation

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.