Description of the digestive tract and feeding habits of the king angelfish and the Cortes angelfish

Morphologically, the digestive tracts of the king angelfish Holacunthus passer and the Cortes angelfish Pomacanthus zonipectus are similar, yet the king angelfish intestine is almost 30% longer than that of the Cortes angelfish. Both pomacanthids have a small mouth with villiform teeth, a short oesophagus, a well-defined stomach, and a terminal sac at the end of the digestive tract. The terminal sac, the acid pH in the stomach, and the long intestine may facilitate efficient use of seaweed nutrients. Stomach contents were analysed to determine diets and interspecific overlap. Seventy-one species were found in the stomachs of the king angelfish and 53 in the stomachs of the Cortes angelfish. Because of the wide range of species in their diets, both angelfish must be regarded as omnivorous. The most frequent foods were seaweed and sponges, but for the king angelfish, crustaceans were also important. A cluster analysis was done to determine whether the diets of these fish were similar by sex, size, or season. No similarities were found. Dietary overlap is high in relation to other pomacanthids.     

Improved two‐stage group sequential procedures for testing a secondary endpoint after the primary endpoint achieves significance

In two‐stage group sequential trials with a primary and a secondary endpoint, the overall type I error rate for the primary endpoint is often controlled by an α‐level boundary, such as an O'Brien‐Fleming or Pocock boundary. Following a hierarchical testing sequence, the secondary endpoint is tested only if the primary endpoint achieves statistical significance either at an interim analysis or at the final analysis. To control the type I error rate for the secondary endpoint, this is tested using a Bonferroni procedure or any α‐level group sequential method. In comparison with marginal testing, there is an overall power loss for the test of the secondary endpoint since a claim of a positive result depends on the significance of the primary endpoint in the hierarchical testing sequence. We propose two group sequential testing procedures with improved secondary power: the improved Bonferroni procedure and the improved Pocock procedure. The proposed procedures use the correlation between the interim and final statistics for the secondary endpoint while applying graphical approaches to transfer the significance level from the primary endpoint to the secondary endpoint. The procedures control the familywise error rate (FWER) strongly by construction and this is confirmed via simulation. We also compare the proposed procedures with other commonly used group sequential procedures in terms of control of the FWER and the power of rejecting the secondary hypothesis. An example is provided to illustrate the procedures.     

Relative efficiency of unequal versus equal cluster sizes in cluster randomized trials using generalized estimating equation models

There is growing interest in conducting cluster randomized trials (CRTs). For simplicity in sample size calculation, the cluster sizes are assumed to be identical across all clusters. However, equal cluster sizes are not guaranteed in practice. Therefore, the relative efficiency (RE) of unequal versus equal cluster sizes has been investigated when testing the treatment effect. One of the most important approaches to analyze a set of correlated data is the generalized estimating equation (GEE) proposed by Liang and Zeger, in which the “working correlation structure” is introduced and the association pattern depends on a vector of association parameters denoted by ρ. In this paper, we utilize GEE models to test the treatment effect in a two‐group comparison for continuous, binary, or count data in CRTs. The variances of the estimator of the treatment effect are derived for the different types of outcome. RE is defined as the ratio of variance of the estimator of the treatment effect for equal to unequal cluster sizes. We discuss a commonly used structure in CRTs—exchangeable, and derive the simpler formula of RE with continuous, binary, and count outcomes. Finally, REs are investigated for several scenarios of cluster size distributions through simulation studies. We propose an adjusted sample size due to efficiency loss. Additionally, we also propose an optimal sample size estimation based on the GEE models under a fixed budget for known and unknown association parameter (ρ) in the working correlation structure within the cluster.     

Spatial and temporal variation in distribution of Gelidium canariensis (Rhodophyta) from natural populations of the Canary Islands

This study was designed to investigate spatial and temporal variation in Gelidium canariensis populations at two shores in northern Gran Canaria during two years. Spatial scales ranged from some hundred meters (distance between shores), 10 to 30 m (distance between plots) to less than 3 m (distance between quadrats). Gelidium individuals were defined as distinct Gelidium clumps. The results show a significant difference in size of clumps between shores, but not on the smaller spatial scales. No significant temporal variation was found. There was no significant temporal or spatial variation in standing crop or density (counts made in quadrats where Gelidium was present, rather than counts for the total shore). Sporophytic and gametophytic clumps were also distinguished by identifying reproductive structures in the field. The total proportion of sporophytes was larger than the proportion of gametophytes, but at a smaller scale there could be a shift in dominance. The survival rate of clumps was similar between shores with a mean survival rate of 85%, but there was a significant difference in recruitment between shores. The results indicate a stable population structure. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative Study of Fatty Acids Profile in Eleven Wild Mushrooms of Boletacea and Russulaceae Families

Eleven species of wild mushrooms which belong to Boletaceae and Russulaceae families were examined by gas chromatography (GC) and gas chromatography–mass spectrometry (GC/MS) analysis for the presence of fatty acids. As far as we know, the fatty acid profiles of B. purpureus and B. rhodoxanthus were described for the first time. Twenty‐six fatty acids were determined. Linoleic (19.5 – 72%), oleic (0.11 – 64%), palmitic (5.9 – 22%) and stearic acids (0.81 – 57%) were present in the highest contents. In all samples, unsaturated fatty acids dominate. Agglomerative hierarchical clustering was used to display the correlation between the fatty acids and their relationships with the mushroom species. Based on the fatty acids profile in the samples, the mushrooms can be divided into two families: Boletaceae and Russulaceae families, using cluster analysis.     

Biosensors based on peptide exposure show single molecule conformations in live cells

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Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry

 Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrometry of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu₁₂MT species. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Addition of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ions whoses masses were consistent with apo-MT, Cu₄MT, Cu₆MT, and Cu₁₂MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu₄ to Cu₁₂MT species. However, formation of the tetracopper MT species remained cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the α or β domain, domain peptides of MT3 were used. Addition of Cu(I) to the apo β domain resulted in a peak consistent with the formation of a four-Cu(I) cluster. This is consistent with reports that Cu(I) ions bind preferentially to the β domain of MTs. Received: 2 June 1998 / Accepted: 21 August 1998     

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.     

Low-temperature magnetic circular dichroism evidence for the conversion of four-iron-sulphur clusters in a ferredoxin from Clostridium pasteurianum into three-iron-sulphur clusters

Oxidation of the 8Fe ferredoxin from Clostridium pasteurianum with potassium ferricyanide, followed by purification on Sephadex G-25 and DE-23 cellulose columns, gives a protein with an intense EPR signal at g 2.01. The low-temperature magnetic circular dichroism (MCD) spectra of this species are different from those of the oxidized high-potential iron protein from Chromatium but identical with the spectra of ferredoxin II from Desulphovibrio gigas. On reduction of the ferricyanide-treated ferredoxin with sodium dithionite only a weak EPR signal with g factors of 2.05, 1.94 and 1.89 is obtained. The low-temperature MCD spectra are strongly temperature dependent with a form similar to those of dithionite-reduced D. gigas ferredoxin II. The MCD magnetization curves are dominated by a species with ground-state effective g factors of $\text{g}{}_{\mathrm{?}}$ 8.0 and g_⊥ 0.0, which are also similar to those determined recently by low-temperature MCD spectroscopy for D. gigas ferredoxin II. The MCD characteristics are quite different from those of dithionite-reduced ferredoxin from Cl. pasteurianum, untreated with ferricyanide. This establishes the close similarity of the iron-sulphur clusters in ferricyanide-treated Cl. pasteurianum ferredoxin and in D. gigas ferredoxin II. The latter is known to contain a single 3Fe centre, similar to that observed in ferredoxin I from Azotobacter vinelandii by X-ray crystallography. Therefore, it is concluded that the [4Fe-4S] clusters of Cl. pasteurianum ferredoxin are converted to 3Fe clusters on oxidation with ferricyanide.     

Variation in Volatile Leaf Oils of Eleven Eucalyptus Species Harvested from Korbous Arboreta (Tunisia)

Hydrodistillation of the dried leaves of eleven species of the genus Eucalyptus L 'Hér ., i.e., E. astringens Maiden , E. camaldulensis Dehnh ., E. diversifolia Bonpl ., E. falcata Turcz ., E. ficifolia F. Muell ., E. gomphocephala DC., E. lehmannii (Schauer ) Benth ., E. maculata Hook ., E. platypus Hook ., E. polyanthemos Schauer, and E. rudis Endl ., harvested from Korbous arboreta (region of Nabeul, northeast of Tunisia) in April 2006, afforded essential oils in yields varying from 0.1±0.1 to 3.8±0.1%, dependent on the species. E. astringens and E. ficifolia showed the highest and the lowest mean percentage of essential oil amongst all the species examined, respectively. Analysis by GC (RI) and GC/MS allowed the identification of 138 components, representing 74.0 to 99.1% of the total oil. The contents of the different samples varied according to the species. The main components were 1,8‐cineole, followed by trans‐pinocarveol ( 1 ), spathulenol ( 2 ), α‐pinene, p‐cymene, (E,E)‐farnesol, cryptone, globulol ( 3 ), β‐phellandrene, α‐terpineol, viridiflorol, and α‐eudesmol. The principal‐component and the hierarchical‐cluster analyses separated the eleven Eucalyptus leaf essential oils into seven groups, each constituting a chemotype.