RNA aptamers selected to bind human immunodeficiency virus type 1 Rev in vitro are Rev responsive in vivo.

RNA aptamers (binding sequences) that can interact tightly and specifically with the human immunodeficiency virus type 1 Rev protein have previously been selected from random sequence pools. Although the selected sequences compete with the wild-type Rev-binding element (RBE) in vitro, it was not known whether they would be able to functionally replace the RBE in vivo. Two aptamers that were different from the wild-type RBE in terms of both primary sequence and secondary structure were inserted into the full-length Rev-responsive element (RRE) in place of the RBE. The hybrid RREs were assayed for their ability to mediate Rev function in vivo using a reporter system. The aptamers were found to be functionally equivalent to the wild-type element when the assay system was saturated with Rev and better than the wild-type element when Rev was limiting. These results demonstrate that the affinity of the primary Rev-binding element rather than its particular sequence may be most responsible for conferring Rev responsiveness on viral mRNAs. Moreover, the fact that increased binding ability can lead to increased Rev responsiveness suggests that cellular factors do not directly influence the Rev:RBE interaction. Finally, since sequences distinct from the RBE are found to be Rev responsive, it may be possible for the RBE to readily mutate in response to drugs or gene therapy reagents that target the Rev:RBE interaction.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Exercise training in childhood-onset systemic lupus erythematosus: a controlled randomized trial

Introduction

Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.

Methods

Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained (TR, n = 10, supervised moderate-intensity aerobic exercise program) and non-trained (NT, n = 9). Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls (C, n = 10) for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise (that is, peak VO₂, chronotropic reserve (CR), and the heart rate recovery (ΔHRR) (that is, the difference between HR at peak exercise and at both the first (ΔHRR1) and second (ΔHRR2) minutes of recovery after exercise).

Results

The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (P > 0.05). In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion (P = 0.01; ES = 1.07), peak speed (P = 0.01; ES = 1.08), peak VO₂ (P = 0.04; ES = 0.86), CR (P = 0.06; ES = 0.83), and in ΔHRR1 and ΔHRR2 (P = 0.003; ES = 1.29 and P = 0.0008; ES = 1.36, respectively) in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis (P > 0.05, TR vs. C). SLEDAI-2K scores remained stable throughout the study.

Conclusion

A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.

Trial registration

NCT01515163.     

Anti-Rex aptamers as mimics of the Rex-binding element

RNA molecules that bind tightly and specifically to a Rex fusion protein have been isolated from a conformationally constrained pool of random sequence RNAs. The anti-Rex aptamers effectively mimic several features of the wild-type Rex-binding element (XBE). The highest-affinity aptamers effectively compete with the wild-type XBE for binding to the RNA-binding domain of Rex, an arginine-rich motif (ARM), but do not bind to the functionally analogous Rev protein or its ARM. However, characteristic sequence and structural motifs found in some of the anti-Rex aptamers may provide insights into how the Rex protein can interact with other viral RNAs, such as the Rev-responsive element. The anti-Rex aptamers can functionally substitute for the XBE in vivo, a result which supports a previously proposed model for mRNA transport in which the viral genome serves as a platform for assembling a nucleoprotein complex that can co-opt the cellular transport apparatus. Overall, these studies suggest that anti-Rex aptamers may serve as RNA decoys of the Rex protein.     

Bioactivity guided isolation of antifungal compounds from the liverwort Bazzania trilobata (L.) S.F. Gray

A dichloromethane and a methanol extract of the liverwort Bazzania trilobata showed antifungal activity against the phytopathogenic fungi Botrytis cinerea, Cladosporium cucumerinum, Phythophthora infestans, Pyricularia oryzae and Septoria tritici. Bioautography on thin-layer chromatograms was used to isolate six antifungal sesquiterpenes: 5- and 7-hydroxycalamenene, drimenol, drimenal, viridiflorol, gymnomitrol and three bisbibenzyls: 6 ',8'-dichloroisoplagiochin C, isoplagiochin D and 6'-chloroisoplagiochin D. Furthermore we report the isolation of gymnomitr-8(12)-en-4-one and the new coumarin 7,8-dihydroxycoumarin-7-O-beta-D-glucuronide. Their structures have been elucidated based on extensive NMR spectral evidence.     

Hepatitis C virus core and nonstructural protein 3 proteins induce pro- and anti-inflammatory cytokines and inhibit dendritic cell differentiation

Antiviral immunity requires recognition of viral pathogens and activation of cytotoxic and Th cells by innate immune cells. In this study, we demonstrate that hepatitis C virus (HCV) core and nonstructural protein 3 (NS3), but not envelope 2 proteins (E2), activate monocytes and myeloid dendritic cells (DCs) and partially reproduce abnormalities found in chronic HCV infection. HCV core or NS3 (not E2) triggered inflammatory cytokine mRNA and TNF-alpha production in monocytes. Degradation of I-kappa B alpha suggested involvement of NF-kappa B activation. HCV core and NS3 induced production of the anti-inflammatory cytokine, IL-10. Both monocyte TNF-alpha and IL-10 levels were higher upon HCV core and NS3 protein stimulation in HCV-infected patients than in normals. HCV core and NS3 (not E2) inhibited differentiation and allostimulatory capacity of immature DCs similar to defects in HCV infection. This was associated with elevated IL-10 and decreased IL-2 levels during T cell proliferation. Increased IL-10 was produced by HCV patients' DCs and by core- or NS3-treated normal DCs, while IL-12 was decreased only in HCV DCs. Addition of anti-IL-10 Ab, not IL-12, ameliorated T cell proliferation with HCV core- or NS3-treated DCs. Reduced allostimulatory capacity in HCV core- and NS3-treated immature DCs, but not in DCs of HCV patients, was reversed by LPS maturation, suggesting more complex DC defects in vivo than those mediated by core or NS3 proteins. Our results reveal that HCV core and NS3 proteins activate monocytes and inhibit DC differentiation in the absence of the intact virus and mediate some of the immunoinhibitory effects of HCV via IL-10 induction.     

Secondary metabolites from the liverwort Jamesoniella colorata

Six new labdane type diterpenoids, three seco-clerodane diterpenoids, jamesoniellide I, along with the two new jamesoniellides K and L, the sesquiterpene waitziacuminone and a new chlorinated bisbibenzyl, 6,6',10,10',12,12'-hexachloroisoperrottetin A, have been isolated from the liverwort Jamesoniella colorata. Their structures were elucidated by NMR spectroscopy. The absolute configuration of 3-oxo-labda-8(17),13(16),14-triene (1) was established by CD spectroscopy.     

Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.