Production and conversion of chitosan with cultures of Mucor rouxii or Phycomyces blakesleeanus

Summary Highly deacetylated chitosan was accumulated in the mycelia ofMucor rouxii orPhycomyces blakesleeanus. These cultures also effected the deacetylation of the chitin ofAspergillus niger mycelium into chitosan. After 96 hours of incubation with these cultures the degree of acetylation of commercial crab shell chitosan was reduced from 25.0% to values between 4.3 and 8.6%. The potential exists for the production of chitosans with tailored physico-chemical properties from waste chitin.     

Group II intron self-splicing. Alternative reaction conditions yield novel products

Reaction parameters were modified to enhance the in vitro reaction rate and to reveal partial and novel reactions of the group II intron 5g of the mitochondrial gene from Saccharomyces cerevisiae encoding cytochrome c oxidase subunit I. One alteration yields separate 5'- and 3'-exons plus linear excised intron as the main products. A linear reaction intermediate, containing intron and 3'-exon, and products resulting from cleavages at two unexpected sites were identified. Spliced exon "reopening," a novel reaction between excised intron and spliced exons, appears responsible for separate 5'- and 3'-exon products.     

Purification of a functional receptor for calcium-channel blockers from rabbit skeletal-muscle microsomes

The dihydropyridine receptor was purified from rabbit skeletal muscle microsomes in the presence of [3H]nitrendipine plus diltiazem or [3H](+)PN 200-110 to an apparent density of 1.5-2 nmol binding sites/mg protein. Sodium dodecyl sulfate gel electrophoresis in the absence of reducing agents yielded three peptide bands of 142, 56 and 30 kDa in a relative ratio of 11:1:1.3, whereas in the presence of 40 mM dithiothreitol bands of 142, 122, 56, 31, 26 and 22 kDa were obtained in a relative ratio of 5.5:2.2:1:0.9:14:0.09. This gel pattern was observed regardless of whether the receptor was purified as a complex with nitrendipine plus diltiazem or with (+)PN 200-110. cAMP-dependent protein kinase phosphorylated preferentially the 142-kDa band up to a stoichiometry of 0.82 +/- 0.07 (15) mol phosphate/mol peptide. The 56-kDa band was phosphorylated only in substoichiometric amounts. [3H]PN 200-110 bound at 4 degrees C to one site with apparent Kd and Bmax values of 9.3 +/- 1.7 nM and 2.2 +/- 0.3 (3) nmol/mg protein, respectively. The binding was stereospecific and was not observed in the presence of 1 mM EGTA. Desmethoxyverapamil interfered with the binding of [3H]PN 200-110 in an apparent allosteric manner. (-)Desmethoxyverapamil inhibited the binding of [3H]PN 200-110 at 37 degrees C and stimulated it at 18 degrees C. In agreement with these results, (-)desmethoxyverapamil increased the dissociation rate of [3H]PN 200-110 from 0.29 min-1 to 0.38 min-1 at 37 degrees C and decreased it threefold from 0.046 min-1 to 0.017 min-1 at 18 degrees C. The (+)isomer of desmethoxyverapamil inhibited PN 200-110 binding at all temperatures tested. d-cis-Diltiazem stimulated the binding of [3H]PN 200-110 at 37 degrees C with an apparent EC50 of 1.4 microM and decreased the dissociation rate from 0.29 min-1 to 0.11 min-1. The stimulatory effect of d-cis-diltiazem was temperature-dependent and was seen only at temperatures above 18 degrees C. These results suggest that the purified dihydropyridine receptor retains the basic properties of the membrane-bound receptor and contains separate sites for at least dihydropyridines and phenylalkylamines.     

Fungal elicitor triggers rapid, transient, and specific protein phosphorylation in parsley cell suspension cultures

Treatment of suspension-cultured parsley (Petroselinum crispum) cells with fungal elicitor triggers rapid, transient and sequential phosphorylation of a number of proteins, as shown by electrophoretic analysis on two-dimensional gels. This response is rapidly reversed by removal of the elicitor from the medium and appears to be specific. It is not observed in cells exposed to other environmental stress factors, such as heat shock, UV irradiation or treatment with mercuric chloride. Pronase digestion of the elicitor has the same negative effect on protein phosphorylation as its previously demonstrated effect on the activation of some pathogen defense-related genes, suggesting a link between these two phenomena. Some of the changes in protein phosphorylation are among the earliest known events following elicitation. The phosphorylation of a neutral 45-kDa protein, which is found in both the microsomal and cytoplasmic fractions, can be observed as early as 1 min after the onset of elicitor treatment. The phosphorylation of a 26-kDa nuclear protein also starts increasing very early. The changes in protein phosphorylation in response to the elicitor are dependent on the presence of Ca2+ in the medium. Our data are compatible with the hypothesis that protein phosphorylation is involved in the signal transduction processes following elicitor recognition by parsley cells.     

The past in short hypercycles

For homogeneous hypercycles with 2, 3 or 4 substances the future behavior of its trajectories is easily understood, in fact any trajectory converges to an equilibrium point as t +. In this paper we study the descent of the trajectories, i.e. their behavior as t -. It turns out that this backward behavior is not as uniform as the forward behavior. In fact, depending on the initial points some -limit sets are singletons while others consist of certain edges of the state simplex.Work partially supported by Deutsche Forschungsgemeinschaft under grant number Au 66–1     

Distinct distribution of CGRP-, enkephalin-, galanin-, neuromedin U-, neuropeptide Y-, somatostatin-, substance P-, VIP- and serotonin-containing neurons in the two submucosal ganglionic neural networks of the porcine small intestine

Summary In addition to differences between the two submucosal ganglionic neural networks, i.e., the plexus submucosus externus (Schabadasch) and the plexus submucosus internus (Meissner), with respect to the occurrence and distribution of serotonin as neurotransmitter, immunocytochemistry also revealed a distinct distribution for various neuropeptides in these two plexuses. Immunoreactivity for galanin, vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P, neuromedin U, enkephalin, somatostatin and neuropeptide Y was found in varicose and non-varicose nerve fibres of both submucosal ganglionic plexuses, albeit with a distinct distributional pattern. The difference in neurotransmitter and/or neuromodulator content between both neural networks became even more obvious when attention was focussed on the immunoreactivity of the nerve cell bodies for these substances. Indeed, neuropeptide Y, enkephalin-and somatostatin-immunoreactive neuronal perikarya as well as serotonergic neuronal cell bodies appear solely in the plexus submucosus externus. Neuromedin U-immunoreactive perikarya, mostly coexisting with substance P, are observed in large numbers in the plexus submucosus internus, whilst they are rare in the plexus submucosus externus. Double-labelling immunostaining for substance P with CGRP and galanin revealed a different coexistence pattern for the two submucosal ganglionic plexuses. The differing chemical content of the neuronal populations supports the hypothesis that the existence of the two submucosal ganglionic plexuses, present in most large mammals including man, not only reflects a morphological difference but also points to differentiated functions.     

Completion of the physical map of Xq28: the location of the gene for L1CAM on the human X Chromosome

The gene for the neural cell adhesion molecule L1 (L1CAM) has been shown to be located close to the color vision pigment genes in mouse and man. This location has been confirmed by a number of different mapping strategies in both species. With pulsed field gel electrophoresis it has been proposed that L1CAM lies between the RCP, GCP, and GDX, G6PD loci. We report here a reinterpretation of the location of this gene, based on the physical linkage of L1CAM to the more proximal locus DXS15. This places L1CAM between this marker and the color vision genes (RCP, GCP), a region very dense in CpG islands, expected to contain a large fraction of the disease genes assigned to the Xq28 region. In combination with the physical mapping data on Xq28 described previously, this closes the last remaining gap in the map of the Xq27–Xq28 region. This removes the last contradiction between the maps of this region in the genomes of man and mouse, and confirms the close similarity of order and distances of markers between these organisms. Offprint requests to: C.M. Disteche     

Downstream DNA sequences are required to activate a gene expressed in the root cortex of embryos and seedlings.

We showed previously that a gene, designated AX92, which is expressed at an early stage of cortex differentiation in the root apex of oilseed rape seedlings, is also expressed in embryos. To compare AX92 gene regulation during embryo-genesis and postembryonic growth, we constructed a chimeric gene consisting of AX92 5' and 3' untranslated and flanking regions fused with a beta-glucuronidase protein coding region. We showed that the chimeric gene is active in both developing cortex cells in the root apical meristems of transgenic oilseed rape seedlings and in cortex cells at the root end of embryonic axes. To determine whether the AX92 gene is regulated by a common mechanism in embryos and seedlings, we analyzed the expression of modified chimeric genes. We showed that the AX92 chimeric gene is regulated combinatorially and that DNA sequences located 3' of the protein coding region are necessary for its activation in the root cortex of both embryos and seedlings. Our results suggest that common regulatory sequences are required to activate the gene in the embryonic and postembryonic root cortex.