In situ generation of iminodiacetic acid groups on nanoporous alumina for the reversible immobilization of enzymes and other biomolecules

Nanoporous alumina membranes were silanized with aminopropylsilane and iminodiacetic acid (IDA) groups were generated in situ by reaction with iodoacetate. The membranes were mounted in standard filter holders, connected to a HPLC system and saturated with selected metal ions. Cu(II) allowed the capture of chicken muscle lactate dehydrogenase with such stability, repeatability and reproducibility that Michaelis–Menten kinetics could be studied. The IDA surface was stable for months and could be depleted and regenerated with metal ions multiple times without appreciable loss of capacity. The binding of lactate dehydrogenase influenced the backpressure to the extent that could be expected for a monolayer according to Poiseuilles law.     

Iodine binding and peroxidase activity in the endostyle of Salpa fusiformis,Thalia democratica,Dolioletta gegenbauri and Doliolum nationalis (Tunicata,Thaliacea)

Summary The protothyroid region in the endostyles of four species of tunicates was examined by means of autoradiography and cytochemistry, at both the light and electronmicroscopic levels. To reveal the primary binding site for iodine, autoradiography was carried out on endostylar tissue from animals that had been incubated with high activity ¹²⁵I^- over a short period of time. The specific iodine binding enzyme, a peroxidase, was traced by its reaction with DAB. In accordance with previous findings, the iodinebinding cells proved to be the same as those containing the peroxidase. There were also strong indications of a secondary uptake of iodinated compounds and subsequent release into the body fluid. Together with the ultrastructural features, the data provided strong evidence indicating that these cells constitute a protothyroid region, which partly functions as an endocrine organ, possibly homologous with the vertebrate thyroid gland. Since the number of zones varied between the species, the numeration of the protothyroid region also varied. However, in all the examined endostyles, the protothyroid region was seen to be situated dorsolaterally to the glandular regions of the endostyle concerned with food capture.     

Localization of the pupil trigger in insect superposition eyes

Summary In the superposition eyes of the sphingid moth Deilephila and the neuropteran Ascalaphus, adjustment to different intensities is subserved by longitudinal migrations of screening pigment in specialized pigment cells. Using ophthalmoscopic techniques we have localized the light-sensitive trigger that controls pigment position.In both species, local illumination of a small spot anywhere within the eye glow of a dark-adapted eye evokes local light adaptation in the ommatidia whose facets receive the light. Details of the response pattern demonstrate that a distal light-sensitive trigger is located axially in the ommatidium, just beneath the crystalline cone, and extends with less sensitivity deep into the clear zone. The distal trigger in Deilephila was shown to be predominantly UV sensitive, and a UV-absorbing structure, presumably the distal trigger, was observed near the proximal tip of the crystalline cone.In Ascalaphus we also found another trigger located more proximally, which causes local pigment reaction in the ommatidia whose rhabdoms are illuminated (the centre of the eye glow). The light-sensitive trigger for this response appears to be the rhabdom itself.     

Purification of synthetic peptides. Immobilized metal ion affinity chromatography (IMAC).

Immobilized metal ion affinity chromatography (IMAC) is a useful method for purification of synthetic peptides with an N-terminal metal-binding amino acid such as His, Trp, or Cys, especially when such residues are not present in other parts of the molecule. In solid phase peptide synthesis (SPPS), capping with acetic anhydride will, in principle, produce truncated peptides as the only side-products due to incomplete couplings. Consequently, only the desired product will carry the affinity label. Most of the impurities, therefore, can be removed by a single passage through an IMAC column. Some representative examples are presented, where fairly large peptides (30-40 amino acid residues) were efficiently purified by this approach.     

Stimulation of some litter-decomposing basidiomycetes by shikimic acid

Shikimic acid, which constitutes 1.5-2.5% of the dry matter in needles of Scots pine, was found to stimulate the growth of various litter-decomposing basidiomycetes of the genera Marasmius, Mycena and Xeromphalina in a synthetic nutrient medium. Out of eighteen litter-decomposing species, ten were stimulated by shikimic acid, wheras the eight mycorrhizal and four wood-rotting species tested were not affected. Maximal effect was obtained at a concentration of ca 2 m M . Growth experiments at varying pH-values indicated active uptake of shikimic acid. Even in the presence of aromatic amino acids, shikimic acid stimulated the growth of the fungi. In certain species the strong inhibiting effect of phenylalanine, tyrosine and tryptophan, when added simultaneously, was reversed in the presence of shikimic acid. Fungi which were stimulated by shikimic acid were also able to use this compound as their sole carbon source. Maximal stimulating effect of shikimic acid occurred when glucose had been added at optimal concentration.     

Mutants with continuous microcycle conidiation in the filamentous fungus Fusarium solani f. sp. pisi

Summary A method of repeated mutation induction and subsequent selection was used to obtain mutants with microcycle conidiation, mc, in Fusarium solani f. sp. pisi. Wild-type, wt, Fusarium has a filamentous (hyphal) growth in a three-stage, conidium—hypha—conidiophore (phialid) vegetative life cycle. The first major mutation induced (by means of the mutagen MNNG) changed the wild-type strain 1–3^- from producing both macro- and microconidia to the mutant strain 19-P2^-, producing only microconidia but preserving the three-stage life cycle. When strain 19-P2^- was exposed to a similar mutagenic treatment mutants without the hyphal stage were obtained. In these mutants the microconidia germinate to form phialids directly, which in turn produce new conidia, thus accomplishing the life cycle as a two-stage, conidium-conidiophore, microcycle. The mc mutants were stabilized after reisolation through back-crossing to the wt of complementary mating type. Mc growth of the mutants is continuous under stable conditions, proceeding uninterrupted to the stationary stage in liquid as well as on solid medium. Mc colonies on solid medium are small and soft, well suited to replication by means of velvet or paper. Large numbers of mature, synchronized conidia are easily obtained from stationary-stage submerged cultures. Sequential mutations are of interest for studies of morphogenetic evolution. The ahyphal mutants make this group of eukaryotes well suited for genetic engineering and biotechnology.     

How signal sequences maintain cleavage specificity

The specificity of the signal sequence cleavage reaction has been postulated to reside in a signal peptidase active site that can bind only to particular (i, i + 2) pairs of amino acids. In this paper, we present further patterns of non-random amino acid utilization in a region around in vivo cleavage sites, and show that they can be interpreted in terms of selection acting to reduce the number of potential competing sites in the vicinity of the correct one.