Multiple xylanases of Cellulomona fimi are encoded by distinct genes

Cellulomonas fimi genomic DNA encoding xylanase activity has been cloned and expressed in Escherichia coli. As judged by DNA hybridization and restriction analysis, twelve xylanase-positive clones carried a minimum of four different xylanase (xyn) genes. The encoded enzymes were devoid of cellulase activity but three of the four bound to Avicel.     

Reconstitution of protein translocation activity from partially solubilized microsomal vesicles

We have used a reconstitution assay to demonstrate that protein translocation activity can be recovered after microsomal vesicles derived from the rough endoplasmic reticulum have been partially solubilized with n-octyl-beta-glucopyranoside. Two independent approaches were used to establish conditions for partially solubilizing microsomal membranes. When the lipid bilayer was disrupted by detergents to the extent that the integrity of the lipid bilayer had been perturbed, membranes were inactive for translocation. However, detergent-treated membranes could be reconstituted in good yield into a translocation competent form once the detergent was removed.     

Structural similarity and distribution of small cryptic plasmids of Lactobacillus curvatus and L. sake

Plasmid profiles of strains of Lactobacillus curvatus and L. sake isolated from meat or sauerkraut were analysed to investigate plasmid homology and distribution in relation to the ecology of these organisms in fermenting foods. A hybridisation probe was constructed by cloning of pLc2, a cryptic, 2.6-kbp plasmid from L. curvatus LTH683, into the Escherichia coli plasmid pRV50. In Southern hybridisations with the digoxygenine labeled pLc2 probe, pLc2-related small plasmids were frequently detected in meat-borne strains of L. casei subsp. pseudoplantarum, L. curvatus, L. sake, L. alimentarius, L. farciminis and L. halotolerans and in L. curvatus and L. sake isolated from sauerkraut. Among 27 Lactobacillus type strains originally isolated from habitats other than meat this type of homology was detected only with plasmids of L. buchneri and L. mali. Restriction-enzyme mapping of six small cryptic plasmids from L. curvatus and L. sake revealed strong structural homology but no similarity to previously characterized plasmids of lactobacilli. The presence of a variable region in addition to a conserved one and the occurrence of deletions during cloning of pLc2 suggest that vectors derived from these plasmids are likely to be structurally unstable.     

Dual action of hydroxylated diphenylethylene estrogens on protein kinase C1

Protein kinase C (PKC) I (gamma), II (beta) and III (alpha) subspecies are all activated by 1,1-di-(p-hydroxyphenyl)ethylene derivatives (DPE) at micromolar concentrations. This PKC activation depends on the presence of both Ca2+ and phosphatidylserine (PS) but does not require diacylglycerol (DG). DPEs enhance PKC activity at low PS concentrations, but not at saturating PS concentrations. Like DG, DPEs increase the apparent affinity of PKC for PS as well as for Ca2+, but lead to a decrease in the catalytic activity (Vmax). In the presence of saturating DG concentrations, DPEs exhibit an inhibitory action. The derivatives also inhibit the activity of the proteolytic fragment of PKC, protein kinase M. It is concluded that DPEs are mixed-type inhibitors, probably interacting with the catalytic domain of the enzyme.     

An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) betaglucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferylbetaglucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.     

Differential effects of anticytoskeletal compounds on the localization and chemical patterns of actin in germinating conidia of Neurospora crassa

Abstract Anti-actin drugs, cytochalasins A and B, inhibited both normal single, and benomyl-induced multiple, germ tube outgrowth from conidia of Neurospora crassa . Actin was cytochemically found to be concentrated in each of the benomyl-induced germ tube tips. No significant quantitative changes either in total actin or its isoforms were measured in the inhibitor-treated germlings. While intact microtubules are required for normal, monopolar axiation of the germ tube, they appear not to be necessary for benomyl-induced multipolar outgrowth which, in contrast, still requires intact actin microfilaments. Microfilaments and microtubules thus play complementary roles in the normal germination of conidia.     

Aminergic neuron systems of lobsters: morphology and electrophysiology of octopamine-containing neurosecretory cells

In the American lobster (Homarus americanus) the biogenic amines serotonin and octopamine appear to play important and opposite roles in the regulation of aggressive behavior, in the establishment and/or maintenance of dominant and subordinate behavioral states and in the modulation of the associated postural stances and escape responses. The octopamine-containing neurosecretory neurons in the thoracic regions of the lobster ventral nerve cord fall into two morphological subgroups, the root octopamine cells, a classical neurohemal group with release regions along second thoracic roots, and the claw octopamine cells, a group that selectively innervates the claws. Cells of both subgroups have additional sets of endings within neuropil regions of ganglia of the ventral nerve cord. Octopamine neurosecretory neurons generally are silent, but when spontaneously active or when activated, they show large overshooting action potentials with prominent after-hyperpolarizations. Autoinhibition after high-frequency firing, which is also seen in other crustacean neurosecretory cells, is readily apparent in these cells. The cells show no spontaneous synaptic activity, but appear to be excited by a unitary source. Stimulation of lateral or medial giant axons, which excite serotonergic cells yielded no response in octopaminergic neurosecretory cells and no evidence for direct interactions between pairs of octopamine neurons, or between the octopaminergic and the serotonergic sets of neurosecretory neurons was found.     

The effects of Bombyx mori silk strain and extraction time on the molecular and biological characteristics of sericin

Sericin was extracted from three strains of Thai Bombyx mori silk cocoons (white shell Chul1/1, greenish shell Chul3/2, and yellow shell Chul4/2) by a high-pressure and high-temperature technique. The characteristics of sericin extracted from different fractions (15, 45, and 60 min extraction process) were compared. No differences in amino acid composition were observed among the three fractions. For all silk strains, sericin extracted from a 15-min process presented the highest molecular weight. The biological potential of the different sericin samples as a bioadditive for 3T3 fibroblast cells was assessed. When comparing sericin extracted from three silk strains, sericin fractions extracted from Chul4/2 improved cell proliferation, while sericin from Chul 1/1 activated Type I collagen production to the highest extent. This study allows the natural variability of sericin obtained from different sources and extraction conditions to be addressed and provides clues for the selection of sericin sources.