Effects of yeast proteinase A, proteinase B and carboxypeptidase Y on yeast phosphofructokinase.

Incubation of a crude yeast extract containing phosphofructokinase with proteinase A, proteinase B or carboxypeptidase Y gave the following results: Proteinase B and carboxypeptidase Y did not change the activity of phosphofructokinase during incubation. On the other hand, incubation with proteinase A resulted in a 40-100% activation; continued incubation, however, led to an inactivation of the enzyme. Addition of allosteric effectors did not change the activation or inactivation process. The activated phosphofructokinase was not changed with respect to pH optimum and ATP inhibition. Molecular weight determination of phosphofructokinase in crude extracts in the presence of inhibitors of proteinase A indicated a molecular weight of 700000. Without inhibitors of proteinase A, the molecular weight was determined to be 600 000, while after 40-100% activation by proteinase A, a molecular weight of 500 000 was obtained. The activity profile of proteinase A in density gradients indicated that this enzyme is bound to variety of cellular proteins.     

Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway

Presenilin 1 (PS1) interacts with telencephalin (TLN) and the amyloid precursor protein via their transmembrane domain (Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Neuron. 32:579-589). Here, we demonstrate that TLN is not a substrate for gamma-secretase cleavage, but displays a prolonged half-life in PS1(-/-) hippocampal neurons. TLN accumulates in intracellular structures bearing characteristics of autophagic vacuoles including the presence of Apg12p and LC3. Importantly, the TLN accumulations are suppressed by adenoviral expression of wild-type, FAD-linked and D257A mutant PS1, indicating that this phenotype is independent from gamma-secretase activity. Cathepsin D deficiency also results in the localization of TLN to autophagic vacuoles. TLN mediates the uptake of microbeads concomitant with actin and PIP2 recruitment, indicating a phagocytic origin of TLN accumulations. Absence of endosomal/lysosomal proteins suggests that the TLN-positive vacuoles fail to fuse with endosomes/lysosomes, preventing their acidification and further degradation. Collectively, PS1 deficiency affects in a gamma-secretase-independent fashion the turnover of TLN through autophagic vacuoles, most likely by an impaired capability to fuse with lysosomes.     

Recent advances in 2D electrophoresis: an array of possibilities

2D electrophoresis is currently the most widespread technique used for performing functional proteomics (i.e., the large-scale analysis of alterations in protein expression levels). Nevertheless, several limitations inherent to this technology have restricted the full potential of this protein differential display methodology for years. This has even led to the abandonment of 2D electrophoresis by several groups that switched to performing gel-free functional proteomics analyses based on liquid chromatography and mass spectrometry. Meanwhile, important recent advances in 2D electrophoresis, such as the introduction of fluorescent 2D difference gel electrophoresis and numerous protein prefractionation techniques, have thoroughly modernized 2D electrophoresis, making it again one of the preferred methods for the analysis of protein expression differences in many laboratories.     

Formation of granules and Methanosaeta fibres in an anaerobic migrating blanket reactor (AMBR)

It has generally been accepted that the formation of granules in anaerobic wastewater treatment systems requires a hydraulic upflow pattern. To evaluate this hypothesis, we operated an anaerobic migrating blanket reactor (AMBR) without a hydraulic upflow pattern, using a synthetic wastewater containing acetate, propionate, butyrate and sucrose. We provided conditions amenable to the formation of granules by operating the system with a moderate hydraulic selection pressure, which in this system was not the result of a hydraulic upflow pattern, but was provided by migration of biomass and intermittent mechanical mixing. Granules were first noticed after 2 months of operation, and it took another 2 months for a mature granular blanket to develop. Besides granules, approximately 1-cm-long Methanosaeta fibres developed and, after 6 months of operation, 30% of biomass consisted of these fibres. Quantitative membrane hybridization showed that almost all the total 16S rRNA extracted from fibres consisted of 16S rRNA from Methanosaeta concilii. This finding indicates that it was possible to develop pockets consisting almost entirely of an organism with a very limited substrate utilization spectrum (only acetate) in a system that was fed a synthetic wastewater containing acetate, propionate, butyrate and sucrose and that is known for its ability to develop biomass with a complex microbial community structure.     

Use of Pentamidine As Secondary Prophylaxis to Prevent Visceral Leishmaniasis Relapse in HIV Infected Patients,the First Twelve Months of a Prospective Cohort Study

Background

Visceral leishmaniasis (VL) has become an important opportunistic infection in persons with HIV-infection in VL-endemic areas. The co-infection leads to profound immunosuppression and high rate of annual VL recurrence. This study assessed the effectiveness, safety and feasibility of monthly pentamidine infusions to prevent recurrence of VL in HIV co-infected patients.

Methods

A single-arm, open-label trial was conducted at two leishmaniasis treatment centers in northwest Ethiopia. HIV-infected patients with a VL episode were included after parasitological cure. Monthly infusions of 4mg/kg pentamidine-isethionate diluted in normal-saline were started for 12months. All received antiretroviral therapy (ART). Time-to-relapse or death was the primary end point.

Results

Seventy-four patients were included. The probability of relapse-free survival at 6months and at 12 months was 79% and 71% respectively. Renal failure, a possible drug-related serious adverse event, occurred in two patients with severe pneumonia. Forty-one patients completed the regimen taking at least 11 of the 12 doses. Main reasons to discontinue were: 15 relapsed, five died and seven became lost to follow-up. More patients failed among those with a CD4+cell count ≤ 50cells/μl, 5/7 (71.4%) than those with counts above 200 cells/μl, 2/12 (16.7%), (p = 0.005).

Conclusion

Pentamidine secondary prophylaxis led to a 29% failure rate within one year, much lower than reported in historical controls (50%-100%). Patients with low CD4+cell counts are at increased risk of relapse despite effective initial VL treatment, ART and secondary prophylaxis. VL should be detected and treated early enough in patients with HIV infection before profound immune deficiency installs.     

Risk Stratification by Self-Measured Home Blood Pressure across Categories of Conventional Blood Pressure: A Participant-Level Meta-Analysis

Background

The Global Burden of Diseases Study 2010 reported that hypertension is worldwide the leading risk factor for cardiovascular disease, causing 9.4 million deaths annually. We examined to what extent self-measurement of home blood pressure (HBP) refines risk stratification across increasing categories of conventional blood pressure (CBP).

Methods and Findings

This meta-analysis included 5,008 individuals randomly recruited from five populations (56.6% women; mean age, 57.1 y). All were not treated with antihypertensive drugs. In multivariable analyses, hazard ratios (HRs) associated with 10-mm Hg increases in systolic HBP were computed across CBP categories, using the following systolic/diastolic CBP thresholds (in mm Hg): optimal, <120/<80; normal, 120–129/80–84; high-normal, 130–139/85–89; mild hypertension, 140–159/90–99; and severe hypertension, ≥160/≥100.Over 8.3 y, 522 participants died, and 414, 225, and 194 had cardiovascular, cardiac, and cerebrovascular events, respectively. In participants with optimal or normal CBP, HRs for a composite cardiovascular end point associated with a 10-mm Hg higher systolic HBP were 1.28 (1.01–1.62) and 1.22 (1.00–1.49), respectively. At high-normal CBP and in mild hypertension, the HRs were 1.24 (1.03–1.49) and 1.20 (1.06–1.37), respectively, for all cardiovascular events and 1.33 (1.07–1.65) and 1.30 (1.09–1.56), respectively, for stroke. In severe hypertension, the HRs were not significant (p≥0.20). Among people with optimal, normal, and high-normal CBP, 67 (5.0%), 187 (18.4%), and 315 (30.3%), respectively, had masked hypertension (HBP≥130 mm Hg systolic or ≥85 mm Hg diastolic). Compared to true optimal CBP, masked hypertension was associated with a 2.3-fold (1.5–3.5) higher cardiovascular risk. A limitation was few data from low- and middle-income countries.

Conclusions

HBP substantially refines risk stratification at CBP levels assumed to carry no or only mildly increased risk, in particular in the presence of masked hypertension. Randomized trials could help determine the best use of CBP vs. HBP in guiding BP management. Our study identified a novel indication for HBP, which, in view of its low cost and the increased availability of electronic communication, might be globally applicable, even in remote areas or in low-resource settings. Please see later in the article for the Editors'' Summary     

Variation in volatile leaf oils of seven eucalyptus species harvested from Zerniza arboreta (Tunisia)

Leaves of seven species of the genus Eucalyptus L'Hér., viz., E. cladocalyx F. Muell., E. citriodora Hook., E. diversicolor F. Muell., E. fasciculosa F. Muell., E. grandis W. Hill, E. ovata Labill., and E. botryoides Sm., were harvested from Zerniza arboreta (region of Sejnene, northwest of Tunisia) in June 2007. Of the latter species, leaves were collected from trees having two origins, Morocco and Italy. Hydrodistillation of the dried leaves provided essential oils in yields varying from 0.4±0.0 to 3.3±0.1%, according to the species. E. citriodora had the highest mean percentage of essential oil amongst the species examined, whereas the lowest one was obtained for E. botryoides originating from Morocco. Analysis by GC (RI) and GC/MS allowed the identification of 140 compounds, representing 92.5 to 99.4% of the total oil composition. The contents of the different samples varied according to the species. The main components were 1,8-cineole (2), followed by α-pinene (1), p-cymene, borneol, α-terpineol, cryptone, spathulenol, trans-pinocarveol (4), bicyclogermacrene (5), caryophyllene oxide, and β-phellandrene. Principal components analysis and hierarchical cluster analysis separated the eight Eucalyptus leaf essential oils into five groups, each constituting a chemotype.     

A Screen-and-Treat Strategy Targeting Visceral Leishmaniasis in HIV-Infected Individuals in Endemic East African Countries: The Way Forward?

In the wake of the HIV epidemic, visceral leishmaniasis (VL), a disseminated protozoan infection caused by the Leishmania donovani complex, has been re-emerging, particularly in North Ethiopia where up to 40% of patients with VL are co-infected with HIV. Management of VL in HIV co-infection is complicated by increased drug toxicity, and high treatment failure and relapse rates with all currently available drugs, despite initiation of antiretroviral treatment. Tackling L. donovani infection before disease onset would thus be a logical approach. A screen-and-treat approach targeting latent or the early stage of infection has successfully been implemented in other HIV-associated opportunistic infections. While conceptually attractive in the context of VL–HIV, the basic understanding and evidence underpinning such an approach is currently lacking. Prospective cohort studies will have to be conducted to quantify the risk of VL in different risk groups and across CD4 cell count levels. This will allow developing clinical prognostic tools, integrating clinical, HIV and Leishmania infection markers. Interventional studies will be needed to evaluate prophylactic or pre-emptive treatment strategies for those at risk, ideally relying on an oral (combination) regimen. Issues like tolerability, emergence of resistance and drug interactions will require due attention. The need for maintenance therapy will have to be assessed. Based on the risk–benefit data, VL risk cut-offs will have to be identified to target treatment to those most likely to benefit. Such a strategy should be complemented with early initiation of antiretroviral treatment and other strategies to prevent HIV and Leishmania infection.