GSE4, a Small Dyskerin- and GSE24.2-Related Peptide,Induces Telomerase Activity,Cell Proliferation and Reduces DNA Damage,Oxidative Stress and Cell Senescence in Dyskerin Mutant Cells

Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.     

The p13 protein of human T cell leukemia virus type 1 (HTLV-1) modulates mitochondrial membrane potential and calcium uptake

Human T cell leukemia virus type 1 (HTLV-1) encodes p13, an 87-amino-acid protein that accumulates in the inner mitochondrial membrane. Recent studies performed using synthetic p13 and isolated mitochondria demonstrated that the protein triggers an inward potassium (K⁺) current and inner membrane depolarization. The present study investigated the effects of p13 on mitochondrial inner membrane potential (Δψ) in living cells. Using the potential-dependent probe tetramethyl rhodamine methyl ester (TMRM), we observed that p13 induced dose-dependent mitochondrial depolarization in HeLa cells. This effect was abolished upon mutation of 4 arginines in p13's α-helical domain that were previously shown to be essential for its activity in in vitro assays. As Δψ is known to control mitochondrial calcium (Ca²⁺) uptake, we next analyzed the effect of p13 on Ca²⁺ homeostasis. Experiments carried out in HeLa cells expressing p13 and organelle-targeted aequorins revealed that the protein specifically reduced mitochondrial Ca²⁺ uptake. These observations suggest that p13 might control key processes regulated through Ca²⁺ signaling such as activation and death of T cells, the major targets of HTLV-1 infection.     

Kinetic and structural features of betaine aldehyde dehydrogenases: Mechanistic and regulatory implications

The betaine aldehyde dehydrogenases (BADH; EC 1.2.1.8) are so-called because they catalyze the irreversible NAD(P)⁺-dependent oxidation of betaine aldehyde to glycine betaine, which may function as (i) a very efficient osmoprotectant accumulated by both prokaryotic and eukaryotic organisms to cope with osmotic stress, (ii) a metabolic intermediate in the catabolism of choline in some bacteria such as the pathogen Pseudomonas aeruginosa, or (iii) a methyl donor for methionine synthesis. BADH enzymes can also use as substrates aminoaldehydes and other quaternary ammonium and tertiary sulfonium compounds, thereby participating in polyamine catabolism and in the synthesis of γ-aminobutyrate, carnitine, and 3-dimethylsulfoniopropionate. This review deals with what is known about the kinetics and structural properties of these enzymes, stressing those properties that have only been found in them and not in other aldehyde dehydrogenases, and discussing their mechanistic and regulatory implications.     

In vitro regeneration of <Emphasis Type="Italic">Lycaste aromatica</Emphasis> (Graham ex Hook) Lindl. (Orchidaceae) from pseudobulb sections

A protocol for in vitro propagation from pseudobulb sections of Lycaste armomatica (Graham ex Hook) Lindl., an ornamental and fragrant orchid, was developed. The effect of four cytokinins: kinetin (K), meta-topolin (mT), N ⁶-benzyladenine (BA), and thidiazuron (TDZ), in equimolar concentrations, was investigated. Shoot formation from apical and basal pseudobulb sections was obtained in all treatments. A few medial sections cultured in media supplemented with BA formed protocorm-like bodies. Shoot formation was greater from the basal section than the apical, and mainly occurred in explants cultured in media containing TDZ. The highest average numbers of shoots per explant were achieved from basal sections cultured in media supplemented with TDZ at 4.4, 8.87 and 2.2 μM, forming on average 9.9, 8.6 and 7.3 shoots per explant, respectively. Since the medial pseudobulb section was the worst explant for propagation of L. aromatica, we recommend that pseudobulbs be divided into two sections; the basal half should be cultured in MS medium supplemented with TDZ at 4.4 μM and the apical half with TDZ at 2.2 μM. Subculturing individual shoots in MS medium without plant growth regulators allows further development and rooting. A survival rate of more than 90% under greenhouse conditions was achieved. This research represents a direct contribution to the conservation and sustainable use of this valuable natural resource.     

Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia

Background

Although some epidemiologic studies found inverse associations between alcohol drinking and Parkinson's disease (PD), the majority of studies found no such significant associations. Additionally, there is only limited research into the possible interactions of alcohol intake with aldehyde dehydrogenase (ALDH) 2 activity with respect to PD risk. We examined the relationship between alcohol intake and PD among Japanese subjects using data from a case-control study.

Methods

From 214 cases within 6 years of PD onset and 327 controls without neurodegenerative disease, we collected information on "peak", as opposed to average, alcohol drinking frequency and peak drinking amounts during a subject's lifetime. Alcohol flushing status was evaluated via questions, as a means of detecting inactive ALHD2. The multivariate model included adjustments for sex, age, region of residence, smoking, years of education, body mass index, alcohol flushing status, presence of selected medication histories, and several dietary factors.

Results

Alcohol intake during peak drinking periods, regardless of frequency or amount, was not associated with PD. However, when we assessed daily ethanol intake separately for each type of alcohol, only Japanese sake (rice wine) was significantly associated with PD (adjusted odds ratio of ≥66.0 g ethanol per day: 3.39, 95% confidence interval: 1.10-11.0, P for trend = 0.001). There was no significant interaction of alcohol intake with flushing status in relation to PD risk.

Conclusions

We did not find significant associations between alcohol intake and PD, except for the daily amount of Japanese sake. Effect modifications by alcohol flushing status were not observed.     

Cyclin levels during TNF-alpha-induced apoptosis in HIV-infected cells.

Cyclins are cell cycle regulatory proteins. We compared the concurrent kinetics of apoptosis and cyclin expression between HIV-infected cells (J1.1), and uninfected Jurkat cells. Cells were cultured with TNF-alpha and harvested at 24, 48 and 72 hr to examine cyclin expression and DNA content. We found a decline in the levels of the mitotic B cyclin in Jurkat cells (16 to 2%, 48 hr), while in J1.1 cells it was observed in cyclin E (60 to 37%, 72 hr). Because cyclin B is mitotic, results suggest that Jurkat cells undergo apoptosis at G2, while J1.1 cells enter mitosis and then die by apoptosis, as no changes in cyclin B or DNA content at G2M were observed. G1 cyclin E decline in J1.1 cells also suggests that they die after entering mitosis. Based on differences in the cyclins involved, it seems that HIV-1 manipulates the cell cycle to protect J1.1 cells from apoptosis induction at G2, a critical cell cycle phase for HIV replication. Thus, cyclins are useful to characterize points in the cell cycle at which apoptosis is induced, and could become excellent tools to evaluate mechanisms of action of antiretroviral drugs in the cell cycle of HIV-infected cells.     

Bcl-2 and Bax exert opposing effects on Ca2+ signaling, which do not depend on their putative pore-forming region

Recent work has shown that Bcl-2 and other anti-apoptotic proteins partially deplete the endoplasmic reticulum (ER) Ca(2+) store and that this alteration of Ca(2+) signaling reduces cellular sensitivity to apoptotic stimuli. We expressed in HeLa cells Bcl-2, Bax, and Bcl-2/Bax chimeras in which the putative pore-forming domains of the two proteins (alpha 5-alpha 6) were mutually swapped, comparing the effects on Ca(2+) signaling of the two proteins and relating them to defined molecular domains. The results showed that only Bcl-2 reduces ER Ca(2+) levels and that this effect does not depend on the alpha 5-alpha 6 helices of this oncoprotein. Soon after its expression, Bax increased ER Ca(2+) loading, with ensuing potentiation of mitochondrial Ca(2+) responses. Then the cells progressed into an apoptotic phenotype (which included drastic reductions of cytosolic and mitochondrial Ca(2+) responses and alterations of organelle morphology). These results provide a coherent scenario that high-lights a primary role of Ca(2+) signals in deciphering apoptotic stimuli.     

Defining Catastrophic Costs and Comparing Their Importance for Adverse Tuberculosis Outcome with Multi-Drug Resistance: A Prospective Cohort Study,Peru

Background

Even when tuberculosis (TB) treatment is free, hidden costs incurred by patients and their households (TB-affected households) may worsen poverty and health. Extreme TB-associated costs have been termed “catastrophic” but are poorly defined. We studied TB-affected households'' hidden costs and their association with adverse TB outcome to create a clinically relevant definition of catastrophic costs.

Methods and Findings

From 26 October 2002 to 30 November 2009, TB patients (n = 876, 11% with multi-drug-resistant [MDR] TB) and healthy controls (n = 487) were recruited to a prospective cohort study in shantytowns in Lima, Peru. Patients were interviewed prior to and every 2–4 wk throughout treatment, recording direct (household expenses) and indirect (lost income) TB-related costs. Costs were expressed as a proportion of the household''s annual income. In poorer households, costs were lower but constituted a higher proportion of the household''s annual income: 27% (95% CI = 20%–43%) in the least-poor houses versus 48% (95% CI = 36%–50%) in the poorest. Adverse TB outcome was defined as death, treatment abandonment or treatment failure during therapy, or recurrence within 2 y. 23% (166/725) of patients with a defined treatment outcome had an adverse outcome. Total costs ≥20% of household annual income was defined as catastrophic because this threshold was most strongly associated with adverse TB outcome. Catastrophic costs were incurred by 345 households (39%). Having MDR TB was associated with a higher likelihood of incurring catastrophic costs (54% [95% CI = 43%–61%] versus 38% [95% CI = 34%–41%], p<0.003). Adverse outcome was independently associated with MDR TB (odds ratio [OR] = 8.4 [95% CI = 4.7–15], p<0.001), previous TB (OR = 2.1 [95% CI = 1.3–3.5], p = 0.005), days too unwell to work pre-treatment (OR = 1.01 [95% CI = 1.00–1.01], p = 0.02), and catastrophic costs (OR = 1.7 [95% CI = 1.1–2.6], p = 0.01). The adjusted population attributable fraction of adverse outcomes explained by catastrophic costs was 18% (95% CI = 6.9%–28%), similar to that of MDR TB (20% [95% CI = 14%–25%]). Sensitivity analyses demonstrated that existing catastrophic costs thresholds (≥10% or ≥15% of household annual income) were not associated with adverse outcome in our setting. Study limitations included not measuring certain “dis-saving” variables (including selling household items) and gathering only 6 mo of costs-specific follow-up data for MDR TB patients.

Conclusions

Despite free TB care, having TB disease was expensive for impoverished TB patients in Peru. Incurring higher relative costs was associated with adverse TB outcome. The population attributable fraction indicated that catastrophic costs and MDR TB were associated with similar proportions of adverse outcomes. Thus TB is a socioeconomic as well as infectious problem, and TB control interventions should address both the economic and clinical aspects of this disease. Please see later in the article for the Editors'' Summary     

Do mosquitoes filter the access of Plasmodium cytochrome b lineages to an avian host?

Many parasites show fidelity to a set of hosts in ecological time but not evolutionary time and the determinants of this pattern are poorly understood. Malarial parasites use vertebrate hosts for the asexual stage of their life cycle but use Dipteran hosts for the sexual stage. Despite the potential evolutionary importance of Dipteran hosts, little is known of their role in determining a parasite's access to vertebrate hosts. Here, we use an avian malarial system in Panama to explore whether mosquitoes act as an access filter that limits the range of vertebrate hosts used by particular parasite lineages. We amplified and sequenced Plasmodium mitochondrial DNA (mtDNA) from Turdus grayi (clay-coloured robin) and from mosquitoes at the same study site. We trapped and identified to species 123 141 female mosquitoes and completed polymerase chain reaction (PCR) screening for Plasmodium parasites in 435 pools of 20 mosquitoes per pool (8700 individuals total) spanning the 11 most common mosquito species. Our primers amplified nine Plasmodium lineages, whose sequences differed by 1.72%–10.0%. Phylogenetic analyses revealed partial clustering of lineages that co-occurred in mosquito hosts. However PAN3 and PAN6, the two primary parasite lineages of T. grayi , exhibited sequence divergence of 8.59% and did not cluster in the phylogeny. We detected these two lineages exclusively in mosquitoes from different genera — PAN3 was found only in Culex (Melanoconion) ocossa , and PAN6 was found only in Aedeomyia squamipennis . Furthermore, each of these two parasite lineages co-occurred in mosquitoes with other Plasmodium lineages that were not found in the vertebrate host T. grayi . Together, this evidence suggests that parasite–mosquito associations do not restrict the access of parasites to birds but instead may actually facilitate the switching of vertebrate hosts that occurs over evolutionary time.     

Low-temperature micronization of a peptide drug in fluid propellant: Case study cetrorelix

Aim of this study was to elaborate an efficient method for the micronization of the decapeptide cetrorelix (a GnRH-antagonist), in order to obtain a microsuspension as basis for other pharmaceutical preparations, such as e.g. inhalation aerosols. A modified pearl-mill coupled with a cryostat was used for the micronization of cetrorelix in fluid propellant and operated under different conditions. The obtained cetrorelix suspensions were analyzed for particle size distribution, purity of cetrorelix, and for metal contamination through abrasion from parts of the mill. The method allowed an effective micronization of cetrorelix. The mean particle size of the initial cetrorelix lyophilizate bulk ware was reduced from 52.5 μ (Volume Mean Diameter, VMD) down to 14.9, 6.1 and 3.1 μm, respectively, respectively. The HPLC analysis of all cetrorelix suspensions after micronization did not show signs of decomposition as compared to the initial product. The elementary analysis of the suspensions performed by inductively coupled plasma mass spectrometry revealed a negligible amount of contaminants in the suspension (Zr=max. 0.6 ppm; Fe, Cr, Ni, Ba, below limit of quantification, i.e.<0.14 ppm). The only appreciable contaminant. Aluminum (Al=1.1 ppm), was derived from the mechanical capping of aluminum canisters prior to analysis. The Zr determination in the suspension of 0.6 ppm, is still considered to be negligible as compared to the legally tolerated limit of air contamination. By low-temperature micronization in fluid propellant, fine drug suspensions of cetrorelix for pMDIs can be directly manufactured in one-step procedure without destruction of the peptide structure and without appreciable product contamination. Published: July 12, 2001.