Di-cluster, seven-iron ferredoxins from hyperthermophilic Sulfolobales

 Seven-iron ferredoxins from the thermoacidophilic archaea Acidianus ambivalens, A. infernus, Metalosphaera prunae and Sulfolobus metallicus were extensively characterised, allowing study of their expression under aerobic and anaerobic growth conditions as well as the putative role in thermal stability of a recently described zinc centre. The archaeon S. metallicus was found to express, under the same growth conditions, two ferredoxins in almost identical amounts, a novelty among Archaea. Most interestingly, these two ferredoxins differ at the N-terminal amino acid sequence in that one has a zinc binding motif (FdA) and the other does not (FdB); in agreement with these findings, FdA contains a zinc ion and FdB does not. These two ferredoxins have identical thermal stabilities, indicating that the zinc atom is not determinant in the protein thermostability. Further, the presence of the additional zinc centre does not interfere with the redox properties of the iron-sulfur clusters since their reduction potentials are almost identical. From the other three archaea, independently of the growth mode in respect to oxygen, only a single zinc-containing ferredoxin was found. EPR studies on the purified proteins, both in the oxidised and dithionite reduced states, allowed the identification of one [3Fe-4S]^1+/0 centre and one [4Fe-4S]^2+/1+ centre in all proteins studied. The complete sequence of A. ambivalens ferredoxin is reported. Together with the data gathered in this study, the properties of the seven-iron ferredoxins from Sulfolobales so far known are re-discussed. Received: 10 June 1998 / Accepted: 25 June 1998     

Internalization of a Bacillus anthracis protective antigen-c-Myc fusion protein mediated by cell surface anti-c-Myc antibodies.

BACKGROUND: Anthrax toxin, secreted by Bacillus anthracis, consists of protective antigen (PA) and either lethal factor (LF) or edema factor (EF). PA, the receptor-binding component of the toxin, translocates LF or EF into the cytosol, where the latter proteins exert their toxic effects. We hypothesized that anthrax toxin fusion proteins could be used to kill virus-infected cells and tumor cells, if PA could be redirected to unique receptors found only on these cells. MATERIALS AND METHODS: To test this hypothesis in a model system, amino acids 410-419 of the human p62(c-myc) epitope were fused to the C-terminus of PA to redirect PA to the c-Myc-specific hybridoma cell line 9E10. RESULTS: The PA-c-Myc fusion protein killed both mouse macrophages and 9E10 hybridoma cells when administered with LF or an LF fusion protein (FP59), respectively. Similar results were obtained with PA, which suggests that PA-c-Myc used the endogenous PA receptor to enter the cells. By blocking the endogenous PA receptors on 9E10 cells with the competitive inhibitor PA SNKEDeltaFF, the PA-c-Myc was directed to an alternate receptor, i.e., the anti-c-Myc antibodies presented on the cell surface. The c-Myc IgG were proven to act as receptors because the addition of a synthetic peptide containing the c-Myc epitope along with PA SNKEDeltaFF further reduced the toxicity of PA-c-Myc + FP59. CONCLUSION: This study shows that PA can be redirected to alternate receptors by adding novel epitopes to the C-terminus of PA, enabling the creation of cell-directed toxins for therapeutic purposes.     

Cutinase purification on poly(ethylene glycol)–hydroxypropyl starch aqueous two-phase systems

The partition behaviour of cutinase on poly(ethylene glycol) (PEG)–hydroxypropyl starch aqueous two-phase systems was characterized. The effect of molecular mass of PEG, the pH of the system and tie-line length on cutinase partition coefficient and cutinase yield to the top phase was investigated for systems prepared with a purified hydroxypropyl starch (Reppal PES 100) and a crude one (HPS). The effect of the presence of different salts, such as sodium chloride, sodium sulphate and ammonium sulphate, on cutinase partition was also studied. The results lead to the conclusion that aqueous two-phase systems composed of PEG and hydroxypropyl starch are not efficient in the purification of cutinase. In the majority of cases, the partition coefficients were very close to 1, with pH being the factor which affects most cutinase partition. Partition coefficients were significantly improved when salts were added to the systems. For PEG 4000–Reppal PES 100 [at pH 4.0; 0.5 M (NH₄)₂SO₄], the partition coefficient for cutinase was 3.7, while a value of 12 was obtained for PEG 4000–HPS (at pH 4.0; 1 M NaCl). An isoelectric point (pI) of 7.8 was confirmed for cutinase by constructing a cross partition graphic from the results obtained in the experiments with different salts.     

Effects of Bothrops asper snake venom on the expression of cyclooxygenases and production of prostaglandins by peritoneal leukocytes in vivo,and by isolated neutrophils and macrophages in vitro

In this study, the ability of Bothrops asper snake venom (BaV) to increase the production of prostaglandins PGE₂ and PGD₂ was assessed in a mouse model in vivo and in inflammatory cells in vitro. In addition, the expressions of COX-1 and COX-2 were assessed. BaV induced an increment in the in vivo synthesis of PGE₂ and PGD₂, together with an enhanced expression of COX-2, but not of COX-1. However, enzymatic activities of COX-1 and COX-2 were increased. Incubation of isolated macrophages and neutrophils with a sub-cytotoxic concentration of BaV in vitro resulted in increased release of PGE₂ and PGD₂ by macrophages and PGE₂ by neutrophils, concomitantly with an increment in the expression of COX-2, but not of COX-1 by both cell types. Our results demonstrate the ability of BaV to promote the expression of COX-2 and to induce the synthesis of proinflammatory prostaglandins. Macrophages and neutrophils may be important targets for this venom under in vivo situation.     

Optimization and Technological Development Strategies of an Antimicrobial Extract from Achyrocline alata Assisted by Statistical Design

Achyrocline alata, known as Jateí-ka-há, is traditionally used to treat several health problems, including inflammations and infections. This study aimed to optimize an active extract against Streptococcus mutans, the main bacteria that causes caries. The extract was developed using an accelerated solvent extraction and chemometric calculations. Factorial design and response surface methodologies were used to determine the most important variables, such as active compound selectivity. The standardized extraction recovered 99% of the four main compounds, gnaphaliin, helipyrone, obtusifolin and lepidissipyrone, which represent 44% of the extract. The optimized extract of A. alata has a MIC of 62.5 μg/mL against S. mutans and could be used in mouth care products.     

Cardiorespiratory Responses and Prediction of Peak Oxygen Uptake during the Shuttle Walking Test in Healthy Sedentary Adult Men

BackgroundThe application of the Shuttle Walking Test (SWT) to assess cardiorespiratory fitness and the intensity of this test in healthy participants has rarely been studied. This study aimed to assess and correlate the cardiorespiratory responses of the SWT with the cardiopulmonary exercise testing (CEPT) and to develop a regression equation for the prediction of peak oxygen uptake (VO2 peak) in healthy sedentary adult men.MethodsIn the first stage of this study, 12 participants underwent the SWT and the CEPT on a treadmill. In the second stage, 53 participants underwent the SWT twice. In both phases, the VO2 peak, respiratory exchange ratio (R), and heart rate (HR) were evaluated.ResultsSimilar results in VO2 peak (P>0.05), R peak (P>0.05) and predicted maximum HR (P>0.05) were obtained between the SWT and CEPT. Both tests showed strong and significant correlations of VO2 peak (r = 0.704, P = 0.01) and R peak (r = 0.737, P<0.01), as well as the agreement of these measurements by Bland-Altman analysis. Body mass index and gait speed were the variables that explained 40.6% (R2 = 0.406, P = 0.001) of the variance in VO2 peak. The results obtained by the equation were compared with the values obtained by the gas analyzer and no significant difference between them (P>0.05) was found.ConclusionsThe SWT produced maximal cardiorespiratory responses comparable to the CEPT, and the developed equation showed viability for the prediction of VO2 peak in healthy sedentary men.     

Comparison of Unplanned Intensive Care Unit Readmission Scores: A Prospective Cohort Study

PurposeEarly discharge from the intensive care unit (ICU) may constitute a strategy of resource consumption optimization; however, unplanned readmission of hospitalized patients to an ICU is associated with a worse outcome. We aimed to compare the effectiveness of the Stability and Workload Index for Transfer score (SWIFT), Sequential Organ Failure Assessment score (SOFA) and simplified Therapeutic Intervention Scoring System (TISS-28) in predicting unplanned ICU readmission or unexpected death in the first 48 hours after discharge from the ICU.MethodsWe conducted a prospective cohort study in a single tertiary hospital in southern Brazil. All adult patients admitted to the ICU for more than 24 hours from January 2008 to December 2009 were evaluated. SWIFT, SOFA and TISS-28 scores were calculated on the day of discharge from the ICU. A stepwise logistic regression was conducted to evaluate the effectiveness of these scores in predicting unplanned ICU readmission or unexpected death in the first 48 hours after discharge from the ICU. Moreover, we conducted a direct accuracy comparison among SWIFT, SOFA and TISS-28 scores.ResultsA total of 1,277 patients were discharged from the ICU during the study period. The rate of unplanned ICU readmission or unexpected death in the first 48 hours after discharge from the ICU was 15% (192 patients). In the multivariate analysis, age (P = 0.001), length of ICU stay (P = 0.01), cirrhosis (P = 0.03), SWIFT (P = 0.001), SOFA (P = 0.01) and TISS-28 (P<0.001) constituted predictors of unplanned ICU readmission or unexpected death. The SWIFT, SOFA and TISS-28 scores showed similar predictive accuracy (AUC values were 0.66, 0.65 and 0.74, respectively; P = 0.58).ConclusionsSWIFT, SOFA and TISS-28 on the day of discharge from the ICU have only moderate accuracy in predicting ICU readmission or death. The present study did not find any differences in accuracy among the three scores.     

4α‐Acetoxyamijidictyol – A New Antifeeding Dolastane Diterpene from the Brazilian Brown Alga Canistrocarpus cervicornis

Chemical investigation of the CH₂Cl₂ crude extract from the brown alga Canistrocarpus cervicornis (Dictyotaceae) led to isolation of one new ( 1 ) and four previously reported dolastane diterpenes ( 2 – 5 ). Their structures were characterized by 1D‐ and 2D‐NMR spectroscopic techniques, including a full single crystal X‐ray diffraction analysis for 1, 2 , and 4 . In addition, the new structure 1 was assayed as chemical defense inhibiting the feeding by the sea urchin Lytechinus variegatus. This study constitutes an additional report broadening the known spectrum of action and defensive roles of secondary metabolites of the C. cervicornis and Dictyotales species.     

Fish Biodiversity of the Vitória-Trindade Seamount Chain,Southwestern Atlantic: An Updated Database

Despite a strong increase in research on seamounts and oceanic islands ecology and biogeography, many basic aspects of their biodiversity are still unknown. In the southwestern Atlantic, the Vitória-Trindade Seamount Chain (VTC) extends ca. 1,200 km offshore the Brazilian continental shelf, from the Vitória seamount to the oceanic islands of Trindade and Martin Vaz. For a long time, most of the biological information available regarded its islands. Our study presents and analyzes an extensive database on the VTC fish biodiversity, built on data compiled from literature and recent scientific expeditions that assessed both shallow to mesophotic environments. A total of 273 species were recorded, 211 of which occur on seamounts and 173 at the islands. New records for seamounts or islands include 191 reef fish species and 64 depth range extensions. The structure of fish assemblages was similar between islands and seamounts, not differing in species geographic distribution, trophic composition, or spawning strategies. Main differences were related to endemism, higher at the islands, and to the number of endangered species, higher at the seamounts. Since unregulated fishing activities are common in the region, and mining activities are expected to drastically increase in the near future (carbonates on seamount summits and metals on slopes), this unique biodiversity needs urgent attention and management.