Structure and Properties of a Complex of α-Synuclein and a Single-Domain Camelid Antibody

The aggregation of the intrinsically disordered protein α-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of α-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to α-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric α-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 bound to a peptide encompassing the nine C-terminal residues of α-synuclein. The NbSyn2:α-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of α-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of α-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of α-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of α-synuclein aggregation in vitro and possibly in vivo.     

Crystallization and identification of a binary complex of a elastase-I and a Carboxypeptidase Aγ from porcine pancreas

A binary complex of elastase I and Carboxypeptidase Aγ has been isolated and crystallized from activated extracts of porcine pancreas. The purification procedure included ammonium sulfate fractionation and autolysis treatment followed by crystallization. The two components in the complex have been separated by DEAE-Sephadex A-50 column chromatography to homogeneity and their identification was demonstrated by NH₂-terminal sequence analysis. An analysis of the reconstitution crystal from the mixtures of both components showed that the complex consisted of a 1:1 molar ratio with elastase I and Carboxypeptidase Aγ, forming a binary complex in crystalline state.     

Synthesis and moisture absorption and retention activities of a carboxymethyl and a quaternary ammonium derivative of α,α-trehalose

Carboxymethyl α,α-trehalose (CMT) and a quaternary ammonium derivative of α,α-trehalose (QT) were successfully prepared, and their moisture absorption and retention activities were assessed. Results showed that both CMT and QT had better moisture absorption abilities at 43% and 81% relative humidity (RH) than α,α-trehalose. In addition, the two α,α-trehalose derivatives had better moisture retention abilities than α,α-trehalose under three humidity conditions: 81% RH, 43% RH, and under dry conditions. Therefore, carboxymethylation and quaternarization could improve the moisture absorption and retention abilities of α,α-trehalose. CMT and QT showed better moisture absorption ability and moisture retention ability than that of hyaluronan (HA), and could potentially find a use as moisture retention ingredient, for example, in cosmetics.     

Cloning and sequencing of inulinase and β-fructofuranosidase genes of a deep-sea Microbulbifer species and properties of recombinant enzymes

An inulinase-producing Microbulbifer sp. strain, JAM-3301, was isolated from a deep-sea sediment. An inulin operon that contained three open reading frames was cloned and sequenced. Two of the three genes were expressed. One product was an endo-inulinase, and the other was a β-fructofuranosidase. Both enzymes worked together to effectively degrade inulin.     

Nonidentity of chromogranin a and dopamine β-monooxygenase

Dopamine β-monooxygenase (EC 1.14.17.1) and chromogranin A from bovine adrenal chromaffin granules were purified by established procedures and examined for evidence of structural identity. The minimum molecular weights were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to be 74,000 and 35,000, respectively. Amino acid analyses of the two proteins are distinct. Dopamine β-monooxygenase does not possess a free amino terminus, whereas chromogranin A has a leucine amino terminus. Analysis in the protein sequencer showed that chromogranin A contains only a single degradable polypeptide chain. Radioactive S-carboxymethyl derivatives of the two proteins were prepared to compare the soluble peptides after thermolysin digestion. These thermolytic peptides were isolated from columns of Dowex 50-X8 resin and both the peptide and radioactive traces revealed no evidence for similarity of the two proteins, either in toto or in part. Thus, although dopamine β-monooxygenase and chromogranin A may sometimes be copurified, they are distinct entities.     

Characterization and comparison of thermostability of purified β-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus

The thermophilic fungus Thermoascus aurantiacus 179-5 and the mesophilic Aureobasidium pullulans ER-16 were cultivated in corn-cob by solid state fermentation for β-glucosidase production. After fermentation both enzymes were purified. The β-glucosidases produced by the strains A. pullulans and T. aurantiacus were most active at pH 4.0–4.5 and 4.5, with apparent optimum temperatures at 80 and 75 °C, respectively. Surprisingly, the enzyme produced by the mesophilic A. pullulans was stable over a wider range of pH (4.5–9.5 against 4.5–6.5) and more thermostable (98% after 1 h at 75 °C against 98% after 1 h at 70 °C) than the enzyme from the thermophilic T. aurantiacus. The t_(1/2) at 80 °C were 90 and 30 min for A. pullulans and T. aurantiacus, respectively. β-Glucosidase thermoinactivation followed first-order kinetics and the energies of denaturation were 414 and 537 kJ mol⁻¹ for T. aurantiacus and A. pullulans, respectively. The result showed that β-glucosidase obtained from the mesophilic A. pullulans is more stable than that obtained from the thermophilic T. aurantiacus.     

Protein and carbohydrate moieties of a preparation of β-lactamase II

1. A crystalline preparation of beta-lactamase II has been separated into two moieties by gel filtration on a column of Sephadex G-100. 2. The first moiety consisted mainly of carbohydrate and showed virtually no beta-lactamase activity. 3. The second moiety was a protein of molecular weight 22500, which was enzymically active. 4. The protein moiety, like the original protein-carbohydrate complex, required Zn(2+) for beta-lactamase activity. It did not differ significantly from the complex in its behaviour to a number of cephalosporin substrates, but was less stable to heat than the complex. 5. About 30% of the total beta-lactamase activity was lost when the protein-carbohydrate complex was separated into the two moieties. This activity was regained when the protein and carbohydrate moieties were mixed, but the mixture did not show the heat stability of the original complex.     

Cloning and Expression of a β-Galactosidase Gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta^®. Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

Cloning and expression of a β-galactosidase gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta(?). Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

Chronicle of a summer and the editing of cinema‐verite

Jean Rouch and Edgar Morin's Chronicle of a Summer is held up as an innovative, seminal “work in the formation of cinéma‐vérité, and has a central position in the development of documentary and ethnographic filmmaking. Although much has been written about fthis film, little attention has been paid to the film's construction. This paper examines ‘elements of the editing structure of Chronicle of a Summer both to consider how this “work was shaped given the constraints of its production and intended reception and to ¦ analyze a relatively neglected aspect of documentary film practice.     

Fumonisins — Importance and occurence of a new group of mycotoxins

This paper describes the importance of fumonisins for human beings and animals and shows data for the occurence in food. Corn-based food samples (n = 299) purchased in the area of munich were analyzed for fumonisin content using an enzyme immunoassay. Fumonisins are mycotoxins produced byFusarium species, especially byFusarium moniliforme andFusarium proliferatum. Occurrence of fumonisins in corn and in cornbased foods and feeds has been reported from almost all over the world. In several animal species different diseases are traced back to fumonisin toxicosis. Fumonisin levels of 5–10 ppm inhorse feed induce “Equine Leucoencephalomalacia” and hepatic lesions. Hepatotoxic (10 150 ppm fumonisin in feed) and pneumotoxic (>150 ppm fumonisin in feed) effects have been reported for swine. Cattle and poultry appear to be less susceptible to fumonisins. Fumonisin B₁ Revels of 50 ppm in the diet of rats cause hepatotoxic and nephrotoxic effects, long time exposure results in hepatic cancer. A possible role of fumonisins in the etiology of human esophageal cancer is under discussion, although no direct causal evidence is known so far. The mode of action of the fumonisins is probably based on inhibition of sphingolipidbiosynthesis caused by the blockade of the enzyme sphyngosine (sphinganine)-N-acyltrans-ferase.     

In search of a functional flora—towards a greater integration of ecology and taxonomy

Large-scale biodiversity informatics projects will not properly address the needs of one important potential user group. Ecologists do not have ready access to datasets which allow them to assign plant species to functional types. We believe that information technology has developed sufficiently to allow taxonomists and ecologists to work together to address this need and develop specimen databases to combine taxonomic data with ecological and ecophysiological information so that this information will be assigned to the correct taxon in the future. Digital images provide a rapid and economical method of vouchering specimen data, reducing the need to store physical vouchers in herbaria.     

Semisynthesis and antiproliferative evaluation of a series of 3′-aminoflavones

A series of 3′-aminoflavones 5,6,7,8-tetra- or 5,7-dioxygenated on the A-ring was synthesized from tangeretin or naringin, two natural Citrus flavonoids. These flavones were evaluated for antiproliferative activity, activation of apoptosis, and inhibition of tubulin assembly. The most antiproliferative flavones exhibit a common 5-hydroxy-6,7,8-trimethoxy substitution pattern on the A-ring.     

A reinterpretation of Na channel gating and permeation in terms of a phase transition between a transmembrane S4 α-helix and a channel-helix

A functional model for the S4/IV -helix of the action potential sodium channel is described by means of a thermodynamic approach. The model is based on a phase transition between the -helix and an ion conducting channel-helix which is similar to the well established helix-coil transition in solution. The right hand channel-helix is a peptide chain with an alternating sequence of torsional angles (¹¹)=(87°, 315°) and (²²)=(22°, 107°) which yields a helix of 13.5 Å per turn. The axial dipole moments of the peptide bonds of this chain of l-amino acids nearly cancel each other out in similar way to those in the gramicidin A channel, which is formed by alternating d-and l-amino acids. The helix, which does not contain any H-bonds, is stabilized by a helical file of water molecules which includes the permeating ion(s). This file turns around the channel-helix to form a relatively stable double helix structure which corresponds to the open channel. Since every third side chain in the S4/IV helix carries a positive charge their environments must be polarized. These polarized regions form a left hand screening-helix around the -helix are broken and the internal -carbon atom is considered as fixed, the outer ten residues leave the membrane while the internal ten residues form the channel-helix. In this configuration every positively charged side chain matches nearly exactly every second polarized region of the screening-helix leaving the three regions in-between exposed to the water file containing the ion(s). This further stabilizes the channel and agrees nicely with the idea of cationic selectivity. An analysis of the energetics of the -helix-channel-helix transition showed that the voltage-independent part of the free energy per helix residue could well be close to 0 kcal/mol and thus be in the range where a transition could occur. Two voltage-dependent contributions were included: the break down of the considerable dipole of the -helix and the outward shift of the positive charges of the side chains upon channel-helix formation. Taking into account the fact that the formation of an -helix is a highly cooperative process the degree of voltage dependence of the probability of formation of a channel-helix proved to be in the same range as experimental values for the open probability of modified Na channels whose inactivation had been removed. With regard to gating currents, the model predicts that 2.74 positive charges are moved in an outward direction. Consequences of the model for other experimental findings are discussed.     

Behaviour of α-aminoadipylcysteine and glutamylcysteine in the presence of intact and disrupted mycelium of a Cephalosporium sp

1. delta-(l-alpha-Aminoadipyl)-l-cysteine, the corresponding d- and dl-alpha-aminoadipyl isomers, delta-(dl-alpha-amino[6-(14)C]adipyl)-l-cysteine and gamma- and alpha-l-glutamyl-l-cysteine were synthesized. 2. The behaviour of delta-(l-aminoadipyl)-l-cysteine and the corresponding d- and dl-alpha-aminoadipyl isomers was studied in the presence of suspensions of intact mycelium of a Cephalosporium sp., suspensions treated ultrasonically and extracts obtained by grinding with alumina. 3. With intact mycelium the l-alpha-aminoadipyl isomer was removed more rapidly from the extracellular fluid than the corresponding d-isomer. 4. Addition of delta-(dl-alpha-amino[6-(14)C]adipyl)-l-cysteine to suspensions of intact mycelium led to the labelling of extracellular and intracellular penicillin N and cephalosporin C, but also to extensive hydrolysis of the dipeptide. 5. Broken-cell systems hydrolysed delta-(l-alpha-aminoadipyl)-l-cysteine and the corresponding d-alpha-aminoadipyl isomer, but the former was hydrolysed more readily than the latter. 6. gamma- and alpha-l-Glutamyl-l-cysteine were also hydrolysed but delta-(l-alpha-aminoadipyl)-l-cysteinyl-l-valine was not. 7. Only part of the enzyme activity in broken-cell systems responsible for the hydrolysis of delta-(alpha-aminoadipyl)-l-cysteine was present in the supernatant obtained on centrifugation at 20000g. 8. Possible implications of these findings are discussed.     

Design and synthesis of a novel pyrrolidinyl pyrido pyrimidinone derivative as a potent inhibitor of PI3Kα and mTOR

Lead optimization efforts that employed structure base drug design and physicochemical property based optimization leading to the discovery of a novel series of 4-methylpyrido pyrimidinone (MPP) are discussed. Synthesis and profile of 1, a PI3Kα/mTOR dual inhibitor, is highlighted.     

Stories and photographs of William A. Arnold (1904–2001), a pioneer of photosynthesis and a wonderful friend

William A. Arnold discovered many phenomena in photosynthesis. In 1932, together with Robert Emerson, he provided the first experimental data that led to the concept of a large antenna and a few reaction centers (photosynthetic unit); in 1935, he obtained the minimum quantum requirement of 8–10 for the evolution of one O₂ molecule; in 1951, together with Bernard L. Strehler, he discovered delayed fluorescence (also known as delayed light emission) in photosynthetic systems; and in 1956, together with Helen Sherwood, he discovered thermoluminescence in plants. He is also known for providing a solid-state picture of photosynthesis. Much has been written about him and his research, including many articles in a special issue of Photosynthesis Research (Govindjee et al. (eds.) 1996); and a biography of Arnold, by Govindjee and Srivastava (William Archibald Arnold (1904–2001), 2014), in the Biographical Memoirs of the US National Academy of Sciences, (Washington, DC). Our article here offers a glimpse into the everyday life, through stories and photographs, of this remarkable scientist.     

Characterization and study of a κ-casein-like chymosin-sensitive linkage

The present report is dealing with the identification, in various unrelated proteins, of protein fragments sharing local sequence and structure similarities with the chymosin-sensitive linkage surrounding the Phe-Met/Ile bond of κ-caseins. In all these proteins, this linkage is observed within an exposed β-strand-like structure, as also predicted for κ-caseins. The structure of one of these fragments, included in glutamine synthetase, particularly superimposes well with the conformation observed for a chymosin inhibitor (CP-113972) within the complex it forms with chymosin and can be similarly accommodated by specificity pockets within the enzyme substrate binding cleft. The effect of the enzyme activity of chymosin was thus tested on glutamine synthetase. Chymosin cut the latter at the Phe-Met linkage, suggesting that this system may locally resemble the κ-casein/chymosin complex.     

Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK _m andk _cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.