Carotenogenesis in Phycomyces

Time course studies of carotenoid production and of mycelial growth in liquid cultures of Phycomyces blakesleeanus wild type [NRRL 1555 (?)], red mutants C9, C10 and C13 and the heterokaryon C2 * C9 are reported. The ratios of the concentrations of lycopene, γ-carotene and β-carotene in the red mutant C13 and in the heterokaryon C2 * C9 during the growth periods were measured. In these strains the concentration of lycopene is close to its final value after 2 days of growth, at a time at which β-carotene is just beginning to be produced. It is suggested that the β-carotene produced late is possibly synthesized via β-zeacarotene.     

Solvent treatment for β-d-galactosidase release from yeast cells

A method is presented for the release of β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from yeast cells. Enzyme release is attained by mixing yeast cells with concentrated solvents (20 to 95%) and subsequently suspending and agitating the cells in buffer. Many solvents, including isopropanol, ethanol and methanol, were found to be effective. Enzyme release into buffer was relatively slow: 10–20 h was required for maximum yields. The release of protease and β-d-galactosidase was monitored. β-d-Galactosidase solubilization was achieved in high yield: 90% of the intracellular enzyme was released into the buffer. Because this method exhibits resistance to yield loss due to microbial degradation and is not sensitive to small changes in solvent in buffer concentration or treatment time, it is particularly suited to industrial-scale enzyme recoveries.     

Immobilization of enzymes on Spheron: 2. β-Amylase — The development of a column reactor—separator

The titanium-chelation method has been used to immobilize β-amylase (1,4-α-d-glucan maltohydrolase, EC 3.2.1.2) on to Spheron. On various grades of Spheron, protein coupling yields of 56–76% were obtained with barley and sweet-potato β-amylases. The specific enzymic activities of the immobilized enzymes fell in the range 3.7–7.6% of those of the soluble enzymes. The immobilized enzymes were more stable than the soluble, especially in the presence of l-cysteine and serum albumin. The presence of cysteine and serum albumin brought about increases in activity in the preparations, presumably by regenerating essential thiol groups in the enzyme which had been oxidized during the operations. Maltose could be separated from amylopectin and other large polysaccharides by chromatography on Spheron P100, and a system was developed in which maltose, produced by hydrolysis of amylopectin applied in pulses to a column of immobilized β-amylase, was separated from starting material and by-products on a second column of Spheron P100.     

Bioelectrochemical fuel cells

In the past 20 years, inorganic fuel cells have been transformed from novelty devices to practical energy transfer-energy storage units. However, the advantage of the high operating efficiency afforded by these fuel cells is partially offset by (a) the limited viability and high cost of the catalysts, (b) the highly corrosive electrolytes, and (c) the elevated operating temperatures. The possibility exists to reduce some of these problems through the development of bioelectrochemical fuel cells. Such biological/electrochemical systems incorporate either microorganisms or enzymes as an active component within the specified electrode compartments. Recent studies with microorganisms as part of the anode compartment have been aimed at defining the mechanism of the observed electrochemical reactions. Recent investigations on the use of cell-free enzyme preparations in the electrode compartments have dealt primarily with developing methodology and defining mechanisms for enhancing the rate of electron transfer from the enzyme-cofactor active site to the solid electrode surface. Applications of this developing technology have been envisioned for analytical chemistry, medical devices, energy transfer, electrochemical synthesis, and detoxification. In this review, the theory and problems of bioelectrochemical fuel cells are described and related to research, both recent and proposed, for the practical development of this area.     

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Evolutionary assembly of cooperating cell types in an animal chemical defense system

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Chemiluminescent Enzyme Immunoassay for Measuring Leptin

Leptin is one of the representative adipocyte-derived protein hormones. Measuring the serum leptin concentration gives an important index for preventing and treating diabetes mellitus and other diseases. We constructed in this study a chemiluminescent enzyme immunoassay (CLEIA) for measuring leptin by using the anti-leptin polyclonal antibody and alkaline phosphatase (ALP). The method applies the IgG-conjugated ferrite particle to capture leptin in a sample and the ALP-conjugated Fab fragment to detect the captured leptin. We tested Block ace, CE510, and bovine serum albumin (BSA) for their abilities to block non-specific binding of ALP-conjugated anti-leptin Fab to the ferrite particle and found BSA to be the most effective. The measurable range with this ELISA for leptin was 0.1–1.0 pg/mL of leptin and the detection limit (blank+2SD) was 0.1 pg/mL of leptin. These results demonstrate sufficient sensitivity with our system to measure the serum leptin concentration and its clinical usefulness. The results also suggest that a sensitive enzyme immunoassay can be constructed by using only one polyclonal antibody.     

Host-selection behaviour by genetically identical aphids with different plant preferences

The behaviour of summer and autumn winged forms of the black bean aphid, Aphis fabae Scopoli (Homoptera: Aphididae), was compared on two plants utilized at different stages of the insect’s life cycle. Adult autumn migrants (gynoparae) are monophagous, colonizing spindle (Euonymus europaeus), whereas polyphagous summer winged aphids (alate virginoparae) are associated with a variety of herbaceous plants, including broad bean (Vicia faba). When aphids from a single clone were given access to a spindle leaf and a bean seedling in choice tests, many virginoparae settled and larviposited on both plant species over 24 h. By contrast, gynoparae showed a clear preference for spindle, with 93.5% of settled adults and 98.3% of larvae on this plant species. Close‐up video monitoring showed that gynoparae discriminated beans from spindle within a 5‐min period, whereas virginoparae behaved similarly on both plant species. For gynoparae, the major behavioural difference on the two plants appeared after a brief (epidermal) stylet penetration, with many insects taking flight within a few seconds of stylet withdrawal from bean. Factors detected during stylet insertion by gynoparae must therefore inhibit take‐off on spindle. Electrical recording experiments showed that aphids often punctured a cell membrane during brief probes on both plant species, and intracellular stylet activities always included a waveform associated with ingestion. When gynoparae puncture spindle cells their behaviour is probably modified by intracellular metabolites detected via gustation of ingested epidermal cell sap. These cues may inhibit the take‐off reflex which otherwise follows probing.