Purification, characterization and biosynthesis of parabutoxin 3, a component of Parabuthus transvaalicus venom.

A novel peptidyl inhibitor of voltage-gated K+ channels, named parabutoxin 3 (PBTx3), has been purified to homogeneity from the venom of Parabuthus transvaalicus. This scorpion toxin contains 37 residues, has a mass of 4274 Da and displays 41% identity with charybdotoxin (ChTx, also called 'alpha-KTx1.1'). PBTx3 is the tenth member (called 'alpha-KTx1.10') of subfamily 1 of K+ channel-blocking peptides known thus far. Electrophysiological experiments using Xenopus laevis oocytes indicate that PBTx3 is an inhibitor of Kv1 channels (Kv1.1, Kv1.2, Kv1.3), but has no detectable effects on Kir-type and ERG-type channels. The dissociation constants (Kd) for Kv1.1, Kv1.2 and Kv1.3 channels are, respectively, 79 microm, 547 nm and 492 nm. A synthetic gene encoding a PBTx3 homologue was designed and expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli. The recombinant protein was purified from the bacterial periplasm compartment using an amylose affinity resin column, followed by a gel filtration purification step and cleavage by factor Xa (fXa) to release the recombinant toxin peptide (rPBTx3). After final purification and refolding, rPBTx3 was shown to be identical to the native PBTx3 with respect to HPLC retention time, mass spectrometric analysis and functional properties. The three-dimensional structure of PBTx3 is proposed by homology modelling to contain a double-stranded antiparallel beta sheet and a single alpha-helix, connected by three disulfide bridges. The scaffold of PBTx3 is homologous to most other alpha-KTx scorpion toxins.     

Isolation and characterization of a novel type of neurotoxic peptide from the venom of the South African scorpion Parabuthus transvaalicus (Buthidae).

The venom of the South African scorpion Parabuthus transvaalicus was characterized using a combination of mass spectrometry and RP-HPLC separation and bioassays. The crude venom was initially separated into 10 fractions. A novel, moderately toxic but very high abundance peptide (birtoxin) of 58 amino-acid residues was isolated, identified and characterized. Each purification step was followed by bioassays and mass spectroscopy. First a C4 RP-HPLC column was used, then a C18 RP Microbore column purification resulted in > 95% purity in the case of birtoxin from a starting material of 230 microg of crude venom. About 12-14% of the D214 absorbance of the total venom as observed after the first chromatography step was composed of birtoxin. This peptide was lethal to mice at low microgram quantities and it induced serious symptoms including tremors, which lasted up to 24 h post injection, at submicrogram amounts. At least seven other fractions that showed different activities including one fraction with specificity against blowfly larvae were identified. Identification of potent components is an important step in designing and obtaining effective anti-venom. Antibodies raised against the critical toxic components have the potential to block the toxic effects and reduce the pain associated with the scorpion envenomation. The discovery of birtoxin, a bioactive long chain neurotoxin peptide with only three disulfide bridges, offers new insight into understanding the role of conserved disulfide bridges with respect to scorpion toxin structure and function.     

Optimum Membrane Structures for Growth of Coliform and Fecal Coliform Organisms

The purpose of this study was to determine the optimum membrane filter structure and characteristics for recovery of coliform organisms. Additionally, other factors such as sterilization method and membrane composition were examined. Fecal coliform growth tests with varied samples indicated that the most critical factor in recovery was surface pore morphology and not other factors previously suspected. Fecal coliform counts showed a dramatic increase, with increasing surface opening sizes. Membrane structures with surface openings large enough to surround the entrapped bacteria are required for optimum growth of fecal coliform organisms. Maximum fecal coliform recoveries are obtained using membranes composed of mixed esters of cellulose exhibiting a surface opening diameter of 2.4 μm and a retention pore size of 0.7 μm.     

Conformational Studies of Australia Antigen by Optical Rotatory Dispersion and Circular Dichroism

The optical rotatory dispersion and circular dichroism of intact, 8 m urea- or sodium dodecyl sulfate-treated, and carbamidomethylated Australia antigen indicated that the antigen possesses a high alpha-helical content similar to human high-density lipoproteins.     

Optimum pH and temperature conditions for xylose fermentation by Pichia stipitis

Pichia stipitis NRRL Y-7124 is a xylose-fermenting yeast able to accumulate ca. 57 g/L ethanol. Because optimum process conditions are important, data were collected to determine the effects of temperature and pH on growth and fermentation rates and product accumulations. Temperatures (26-35 degrees C) providing optimum biomass and ethanol productivities did not necessarily provide maximum ethanol accumulation. Xylitol and residual xylose concentrations increased with temperature. Maximum ethanol selectivity was achieved at 25-26 degrees C with minimal sacrifice to production rates. The temperature optimum for xylose could not be generalized to glucose fermentations, in which ethanol productivity and accumulation were optimum at 34 degrees C. The optimum pH range for growth and fermentation on xylose was 4-7 at 25 degrees C.     

Mechanism of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. Structures of complexes with the substrate

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniae hyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-A resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn(349), His(399), and Tyr(408), is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the alpha- and beta-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme.     

The Crystal Structures of Yeast Get3 Suggest a Mechanism for Tail-Anchored Protein Membrane Insertion

Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a “finger” domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an “open” conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a “closed” conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.     

Structures of the Human Poly (ADP-Ribose) Glycohydrolase Catalytic Domain Confirm Catalytic Mechanism and Explain Inhibition by ADP-HPD Derivatives

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5′-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.     

A Prototype of a Biomimetic Mantle Jet Propeller Inspired by Cuttlefish Actuated by SMA Wires and a Theoretical Model for Its Jet Thrust

The cuttlefish have higher swimming speed and more maneuverability than most of the fish mainly benefiting from their unique jet propulsion mechanism, which is realized by the contraction and expansion of their flexible mantle. However it is difficult to mimic this jet propulsion mechanism using conventional electro-mechanical structures. In this paper, the musculature of the cuttlefish mantle and how the mantle flexibly contracts and expands were analyzed first. Then the Shape Memory Alloy（SMA） wires were chosen as the actuators and the soft silica gel was chosen as the body materials to develop a biomimetic mantle jet propeller. The SMA wires were embedded within the soft silica gel formed with cuttlefish mantle shape along the annular direction to mimic the circular muscles of cuttlefish mantle. The water was squeezed out the mantle cavity to form rear jets when the biomimetic mantle was contracted by SMA wires. A mechanical model and a thermal model were established to analyze the jet thrust and the jetting frequency. Theoretical analysis shows that the jet thrust is proportional to the square of the rate of change of SMA strain. Increasing the driving voltage can improve the rate of change of SMA strain, thus can improve both the jet thrust and the jetting frequency. However the j etting frequency is mainly restricted by the cooling of SMA wires. To maximize the jetting frequency, the optimal driving parameters for different driving voltage were calculated. The propulsion performance was tested and the results show that the jet thrust can increase with the driving voltage as predicted and the maximum average jet thrust is 0.14 N when the driving voltage is 25 V. The swimming test was carried out to verify the feasibility of the novel design. It is shown that the biomimetic jet propeller can swim with higher speed as the jet thrust and jetting frequency increase and the maximum speed can reach 8.76 cm·s^-1 （0.35 BL·s^-1） at a jetting frequency of 0.83 Hz.     

Time and Site of Inoculation of Maize for Optimum Infection of Ears by Stenocarpella maydis

The optimum inoculation position on the maize plant, and optimum time of inoculation with Stenocarpella maydis for artificial induction of ear rot were determined in field trials. The placement of a conidial suspension on the shank at silking resulted in the highest incidence of S. maydis ear rot.     

Screening for Tannin Degradation by Rumen and Faecal Samples of Wild and Domestic Animals in Ethiopia

Summary Tannins limit the use of fodder trees as feed for ruminants. Removal of the effects of tannins would thus improve the nutritional quality of these trees. This prompted the study to evaluate the effect of rumen or faecal mixed cultures from different animals on tannin degradation. Tannin extracts, tannic acid and gallic acid were used to enrich media to assess if rumen or faecal mixed cultures could degrade the phenolic compounds. Rumen fluid of Acacia-adapted sheep, sheep fed on wheat bran, bush duikers (Sylvicapra grimmia) and goats fed on Leucaena pallida and Sesbania goetzei were separately inoculated into Growth Study Medium (GSM) and incubated for 5-15 days. Faecal samples from dikdik (Madoqua guentheri), camel (Camelus dromedarius), zebra (Equus quagga), Grant’s gazelle (Gazella granti) and hartebeest (Alcelaphus buselaphus) were also separately inoculated into GSM media and incubated from 3-5 days. TLC results showed that mixed cultures from rumen fluids of Acacia-adapted sheep, sheep on wheat bran, goats on Leucaena pallida and Sesbania goetzei partially hydrolysed tannic acid to pyrogallol. Complete degradation of the heterocyclic ring in tannic acid and gallic acid was achieved by the mixed cultures from the faecal samples of dikdik and this was confirmed by HPLC. Mixed cultures from faecal samples of camel hydrolysed gallic acid to phloroglucinol. This study has demonstrated that faecal microorganisms of Ethiopian dikdik could completely degrade hydrolysable tannin.