A simple apparatus for controlling nucleation and size in protein crystal growth

A simple device is described for controlling vapor equilibrium in macromolecular crystallization as applied to the protein crystal growth technique commonly referred to as the "hanging drop" method. Crystal growth experiments with hen egg white lysozyme have demonstrated control of the nucleation rate. Nucleation rate and final crystal size have been found to be highly dependent upon the rate at which critical supersaturation is approached. Slower approaches show a marked decrease in the nucleation rate and an increase in crystal size.     

The Alacoil: A very tight,antiparallel coiled-coil of helices

The Alacoil is an antiparallel (rather than the usual parallel) coiled-coil of α-helices with Ala or another small residue in every seventh position, allowing a very close spacing of the helices (7.5–8.5 Å between local helix axes), often over four or five helical turns. It occurs in two distinct types that differ by which position of the heptad repeat is occupied by Ala and by whether the closest points on the backbone of the two helices are aligned or are offset by half a turn. The aligned, or ROP, type has Ala in position “d” of the heptad repeat, which occupies the “tip-to-tip” side of the helix contact where the Cα–Cβ bonds point toward each other. The more common offset, or ferritin, type of Alacoil has Ala in position “a” of the heptad repeat (where the Cα-Cβ bonds lie back-to-back, on the “knuckle-touch” side of the helix contact), and the backbones of the two helices are offset vertically by half a turn. In both forms, successive layers of contact have the Ala first on one and then on the other helix. The Alacoil structure has much in common with the coiled-coils of fibrous proteins or leucine zippers: both are α-helical coiled-coils, with a critical amino acid repeated every seven residues (the Leu or the Ala) and a secondary contact position in between. However, Leu zippers are between aligned, parallel helices (often identical, in dimers), whereas Alacoils are between antiparallel helices, usually offset, and much closer together. The Alacoil, then, could be considered as an “Ala anti-zipper.” Leu zippers have a classic “knobs-into-holes” packing of the Leu side chain into a diamond of four residues on the opposite helix; for Alacoils, the helices are so close together that the Ala methyl group must choose one side of the diamond and pack inside a triangle of residues on the other helix. We have used the ferritin-type Alacoil as the basis for the de novo design of a 66-residue, coiled helix hairpin called “Alacoilin.” Its sequence is: cmSP DQWDKE A AQYDAHA QE FEKKS HRNng TPEA DQYRHM A SQY QAMA QK LKAIA NQLKK Gseter (with “a” heptad positions underlined and nonhelical parts in lowercase), which we will produce and test for both stability and uniqueness of structure.     

Diel variations of selected physico-chemical parameters in Lake Kissimmee,Florida

Every two months, diel depth profiles were made of dissolved oxygen, pH, temperature and conductivity at two stations in Lake Kissimmee, Florida, from July 1974 through June 1975. Results suggest that stratification does not occur in the large (137 km²), shallow (mean depth 2.5 m) lake, though steep vertical gradients in these parameters may develop. The data also suggest that when these gradients do occur, they rapidly degenerate at night or under slight wind stress. Since many Florida lakes are relatively shallow (mean depth < 5 m) and topography offers little obstruction to wind action, it is believed that the holomictic condition observed in Lake Kissimmee may be typical of the state's lakes, particularly those in the central portion of the peninsula.     

Structural features of paramyxovirus F protein required for fusion initiation

On the basis of the coordinates of the related Newcastle disease virus (NDV) F protein, Valine-94, a determinant of measles virus (MV) cytopathicity, is predicted to lie in a cylindrical cavity with 10 A diameter located at the F neck. A 16-residue domain around V94 is functionally interchangeable between NDV and MV F, supporting our homology model. Features of the cavity are conserved within the Paramyxovirinae. A hydrophobic base and a hydrophilic residue at the rim are required for surface expression. Small residue substitutions predicted to open the cavity were found to disrupt transport or limit fusogenicity of transport-competent mutants but can be compensated for by simultaneous insertion of larger residues at the opposing wall. Variants containing histidine substitutions mediate fusion at pH 8.5, while at pH 7.2 fusion is blocked, suggesting that functionality requires low charge in the cavity. These results indicate that specific structural features of the cavity are essential for paramyxovirus fusion initiation.     

Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv

Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.     

Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola

The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola. The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes. The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas). One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria. The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.     

Fusogenic variants of a noncytopathic paramyxovirus

SER virus is a type 5 parainfluenza virus that does not exhibit syncytium formation, in contrast to most other paramyxoviruses. This property has been attributed, at least in part, to the presence of an extension of the cytoplasmic tail (CT) of the SER F protein, as truncations or mutations of this region resulted in enhanced fusion. In this study we used repeated passage to select for mutant SER viruses, which were found to be fusogenic. The mutant viruses replicated at levels comparable to or higher than the wild-type SER virus and caused plaque formation, in contrast to the wild-type virus which does not form plaques. The mutants differed strikingly in their plaque sizes. The F genes of mutant viruses were cloned and sequenced and shared some mutations, including a proline-to-leucine change at position 22 and an isoleucine-to-leucine substitution at position 191; other changes that were specific to each mutant were also found. The HN proteins of mutant viruses also showed mutations spanning the length of the protein whereas the M protein showed a consistent mutation, threonine to isoleucine, at position 129. The structure of the F protein was used to identify residues involved in the mutant phenotypes in terms of their location and proximity to heptad repeat domains.     

UNC-98 and UNC-96 interact with paramyosin to promote its incorporation into thick filaments of Caenorhabditis elegans

Mutations in unc-96 or -98 cause reduced motility and a characteristic defect in muscle structure: by polarized light microscopy birefringent needles are found at the ends of muscle cells. Anti-paramyosin stains the needles in unc-96 and -98 mutant muscle. However there is no difference in the overall level of paramyosin in wild-type, unc-96, and -98 animals. Anti-UNC-98 and anti-paramyosin colocalize in the paramyosin accumulations of missense alleles of unc-15 (encodes paramyosin). Anti-UNC-96 and anti-UNC-98 have diffuse localization within muscles of unc-15 null mutants. By immunoblot, in the absence of paramyosin, UNC-98 is diminished, whereas in paramyosin missense mutants, UNC-98 is increased. unc-98 and -15 or unc-96 and -15 interact genetically either as double heterozygotes or as double homozygotes. By yeast two-hybrid assay and ELISAs using purified proteins, UNC-98 interacts with paramyosin residues 31-693, whereas UNC-96 interacts with a separate region of paramyosin, residues 699-798. The importance of surface charge of this 99 residue region for UNC-96 binding was shown. Paramyosin lacking the C-terminal UNC-96 binding region fails to localize throughout A-bands. We propose a model in which UNC-98 and -96 may act as chaperones to promote the incorporation of paramyosin into thick filaments.     

Efficient and Stable TiO2:Pt–Cu(In,Ga)Se2 Composite Photoelectrodes for Visible Light Driven Hydrogen Evolution

Novel thin film composite photocathodes based on device‐grade Cu(In,Ga)Se₂ chalcopyrite thin film absorbers and transparent conductive oxide Pt‐implemented TiO₂ layers on top are presented for an efficient and stable solar‐driven hydrogen evolution. Thin films of phase‐pure anatase TiO₂ are implemented with varying Pt‐concentrations in order to optimize simultaneously i) conductivity of the films, ii) electrocatalytic activity, and iii) light‐guidance toward the chalcopyrite. Thereby, high incident‐photon‐to‐current‐efficiencies of more than 80% can be achieved over the full visible light range. In acidic electrolyte (pH 0.3), the most efficient Pt‐implemented TiO₂–Cu(In,Ga)Se₂ composite electrodes reveal i) photocurrent densities up to 38 mA cm^?2 in the saturation region (?0.4 V RHE, reversible hydrogen electrode), ii) 15 mA cm^?2 at the thermodynamic potential for H₂‐evolution (0 V RHE), and iii) an anodic onset potential shift for the hydrogen evolution (+0.23 V RHE). It is shown that the gradual increase of the Pt‐concentration within the TiO₂ layers passes through an efficiency‐ and stability‐maximum of the device (5 vol% of Pt precursor solution). At this maximum, optimized light‐incoupling into the device‐grade chalcopyrite light‐absorber as well as electron conductance properties within the surface layer are achieved while no degradation are observed over more than 24 h of operation.     

Detection of hemolytic bacteria from <Emphasis Type="Italic">Palythoa caribaeorum</Emphasis> (Cnidaria,Zoantharia) using a novel palytoxin-screening assay

Palytoxin (PTX), one of the most potent and chemically complex marine toxins, is predominantly found in zoanthid corals and sporadically in dinoflagellates. Its biosynthesis and metabolic pathways are largely unknown. However, the widespread occurrence of the toxin in phylogenetically distinct marine organisms is consistent with its production by microorganisms and subsequent accumulation in the food chain. To investigate a possible microbial origin, bacteria from two zoanthid corals (Palythoa caribaeorum, Zoanthus pulchellus) and one sponge (Neofibularia nolitangere) were isolated. More than 250 bacteria were screened for hemolysis using a newly developed PTX-screening assay of which 7% showed PTX-like hemolytic activity. 16S rRNA gene sequencing revealed that these bacterial isolates belonged to strains of Bacillus cereus group (n = 11) as well as the genera Brevibacterium (n = 4) and Acinetobacter (n = 2). The results indicate the presence of Na⁺/K⁺-ATPase toxins and possibly PTX in hemolytic bacteria from P. caribaeorum.