HTLV-I antisense transcripts initiating in the 3'LTR are alternatively spliced and polyadenylated

BACKGROUND: Antisense transcription in retroviruses has been suggested for both HIV-1 and HTLV-I, although the existence and coding potential of these transcripts remain controversial. Thorough characterization is required to demonstrate the existence of these transcripts and gain insight into their role in retrovirus biology. RESULTS: This report provides the first complete characterization of an antisense retroviral transcript that encodes the previously described HTLV-I HBZ protein. In this study, we show that HBZ-encoding transcripts initiate in the 3' long terminal repeat (LTR) at several positions and consist of two alternatively spliced variants (SP1 and SP2). Expression of the most abundant HBZ spliced variant (SP1) could be detected in different HTLV-I-infected cell lines and importantly in cellular clones isolated from HTLV-I-infected patients. Polyadenylation of HBZ RNA occurred at a distance of 1450 nucleotides downstream of the HBZ stop codon in close proximity of a typical polyA signal. We have also determined that translation mostly initiates from the first exon located in the 3' LTR and that the HBZ isoform produced from the SP1 spliced variant demonstrated inhibition of Tax and c-Jun-dependent transcriptional activation. CONCLUSION: These results conclusively demonstrate the existence of antisense transcription in retroviruses, which likely plays a role in HTLV-I-associated pathogenesis through HBZ protein synthesis

Satellite Cells Senescence in Limb Muscle of Severe Patients with COPD

Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Québec, Canada Rationale: The maintenance of peripheral muscle mass may be compromised in chronic obstructive pulmonary disease (COPD) due to premature cellular senescence and exhaustion of the regenerative potential of the muscles. Methods: Vastus lateralis biopsies were obtained from patients with COPD (n = 16) and healthy subjects (n = 7). Satellite cell number and the proportion of central nuclei, as a marker of muscle regenerative events, were assessed on cryosections. Telomere lengths, used as a marker of cellular senescence, were determined using Southern blot analyses. Results: Central nuclei proportion was significantly higher in patients with COPD with a preserved muscle mass compared to controls and patients with COPD with muscle atrophy (p,0.001). In COPD, maximal telomere length was significantly decreased compared to controls (p,0.05). Similarly, minimal telomere length was significantly reduced in GOLD III–IV patients with muscle atrophy compared to controls (p,0.005). Minimal, mean and maximum telomere lengths correlated with mid-thigh muscle cross-sectional area (MTCSA) (R = 0.523, p = 0.005; R = 0.435, p = 0.019 and R = 0.491, p = 0.009, respectively). Conclusions: Evidence of increased regenerative events was seen in GOLD III–IV patients with preserved muscle mass. Shortening of telomeres in GOLD III–IV patients with muscle atrophy is consistent with an increased number of senescen

Study of the Relation between Hypoxia and Muscle Atrophy

Background : Skeletal muscle atrophy is an important feature of chronic obstructive pulmonary disease (COPD) and it is recognized to have considerable clinical impacts. Unfortunately, factors contributing to muscle wasting in COPD are poorly understood. Hypoxemia is typical in COPD and several evidences link hypoxic conditions and protein breakdown. We propose that hypoxia participate to muscle atrophy by increasing Ubiquitin-Proteasome (UP) system activity and by decreasing the activity of IGF/PI3K/Akt synthesis pathway. Methods: To test this hypothesis, L6 muscle myotubes were either exposed to hypoxia (1% O2) or normoxia (21% O2). Results: After 24 hours of hypoxic exposure, we found a significant rise in the chymotrypsin and caspase-like 20S proteasome activities. Proteolysis was confirmed by an accumulation of a 14 kDa actin fragment during hypoxia. An elevation of Atrogin-1 mRNA expression was also observed in similar conditions. A decline in Akt phosphorylation was noticed in hypoxia. These changes were attenuated by insulin treatment. Conclusion: Proteolysis is accentuated in myotubes exposed to hypoxia and the UP system appears to be involved. In addition, protein synthesis seems to be affected as a lower Akt activity was observed. However, the IGF/PI3K/Akt pathway can still be stimulated by a suitable signal suggesting that therapies targeting this pathway are conceivable

Central nucleus quantification.

<p><b>A</b>) Cryosections were analyzed by labeling nuclei (blue) and laminin (red). Immuno-detection is shown for 10 µm skeletal muscle cryosections. The number of central nuclei (white arrows) is expressed over 100 fibres and reported as a ratio. Ratio are calculated from 9 COPD and MTCSA >70 cm², 7 COPD and MTCSA <70 cm² and 7 healthy subjects. <b>B</b>) The total numbers of central nuclei were quantified per 100 fibres for patients with COPD and MTCSA >70 cm², patients with COPD and MTCSA <70 cm² and for healthy subjects. Nucleus inside the perimeter of a given fibre delimited by laminin was counted as a central nucleus, expressed over 100 fibres and reported as a ratio. Distinct letter represents a statistically significant difference (ANOVA; p<0.05).</p

Telomere length.

<p><b>A</b>) Minimal, <b>B</b>) Mean and <b>C</b>) Maximal telomere restriction fragment length of the <i>vastus lateralis</i> plotted in healthy subjects (n = 7), patients with COPD and MTCSA >70 cm²) (n = 9) and patients with COPD and MTCSA <70 cm² (n = 7). The solid horizontal line represents the mean value for the group. Distinct letter represents a statistically significant difference (ANCOVA; p<0.05).</p

Satellite cells quantification.

<p><b>A</b>) <i>Vastus lateralis</i> muscle cryosections were labeled for co-expression and localization of nuclear Paired box transcription factor 7 (green), DAPI (blue) and laminin (red). Cells positive for pax7 or NCAM (not shown) and DAPI located between the plasmalemma and the basal lamina were counted as satellite cells (white arrow). The number of satellite cells is expressed over 100 fibres and reported as a ratio. Ratio are calculated from 9 COPD and MTCSA >70 cm², 7 COPD and MTCSA <70 cm² and 7 healthy subjects. <b>B</b>) The numbers of nuclei having a positive label for Pax7 (white) and NCAM (black) were counted as satellite cells. Cells positive for pax7 or NCAM and DAPI located between the plasmalemma and the basal lamina were counted as satellite cells. The number of satellite cells is expressed over 100 fibres and reported as a ratio; ANOVA; p>0.05.</p

Subjects characteristics

<p>Definition of abbreviations: BMI = Body mass index; FEV₁ = Forced expiratory volume in 1 second; FVC = Forced vital capacity; DLCO = Diffusing capacity of carbon monoxide; MTCSA = Mid-thigh cross-sectional area; CSA = Cross-sectional area. Values with different lower case letter are significantly different (p<0.05).</p>*<p>p<0.001;</p>†<p>p<0.01.</p

Minimal, mean and maximum telomere length correlation with MTCSA.

<p><b>A</b>) Relationship between minimal telomere length and Mid-thigh Cross-Sectional Area (MTCSA) in patients with COPD and healthy controls. Telomere length significantly correlated with MTCSA in the studied population (r = 0.523; p = 0.005). <b>B</b>) Relationship between mean telomere length and Mid-thigh Cross-Sectional Area (MTCSA) in patients with COPD and healthy controls. Telomere length significantly correlated with MTCSA in the studied population (r = 0.435; p = 0.019). <b>C</b>) Relationship between maximum telomere length and Mid-thigh Cross-Sectional Area (MTCSA) in patients with COPD and healthy controls. Telomere length significantly correlated with MTCSA in the studied population (r = 0.491; p = 0.009).</p