Production of extracellular enzymes by Thermomonospora curvata during growth on protein-extracted lucerne fibres

After extraction of food protein from lucerne, the residual fibre was used as a carbon and energy source by the thermophilic actinomycete, Thermomonospora curvata. Induction of catabolic exoenzymes during growth for 7 d on the fibre at 53°C in a mineral salts minimal medium was compared with that on a variety of other inductive substrates. A fibre concentration of 1.5% (w/v) was optimal for total protein secretion. The fibre was a poor substrate for amylase production due to lack of inducer rather than to catabolite repression by soluble sugars released during degradation. β-Glucosidase release during growth on the fibre was about 10 times that observed in cultures grown on cellobiose or cellulose, but production of other cellulolytic enzymes was about one-half that produced on cellulose. Pectinolytic activity (measured as polygalacturonate lyase) was equal to that produced on pectin. Cells grown on the fibre released about eight times as much proteinase as those grown on cellulose, but proteolytic activity was transient and decreased rapidly during later growth. Xylanase appeared to be co-ordinately induced with cellulolytic enzymes; comparable maximal activities, observed during growth on either the fibre or cellulose, were three times that produced on xylan or xylose.     

Size and shape of the multicatalytic proteinase from rat skeletal muscle

The multicatalytic proteinase from rat skeletal muscle, a non-lysosomal high molecular weight enzyme active at neutral to alkaline pH, has been examined in the electron microscope as well as by dynamic laser light scattering. Both methods reveal monodisperse particles. Electron micrographs show a cylinder-shaped complex with a diameter of 11 nm and a length of 16 nm in negatively stained, and a diameter of 9.6 nm and a length of 14.3 nm in freeze-dried, heavy metal replicated specimens. The molecule is composed of four rings or disks.     

Inhibition of Swarming of Proteus by Sodium Tetradecyl Sulfate, β-Phenethyl Alcohol,and p-Nitrophenylglycerine

The effects of sodium tetradecyl sulfate (STS), β-phenethyl alcohol (PEA), and p-nitrophenylglycerol (PNPG) on motility, swarming, flagellation, and growth of Proteus were examined. Growth-inhibitory concentrations (GIC) and swarming-inhibitory concentrations (SIC) were determined. A characterization of the swarming-inhibitory efficacy of these compounds was based on their GIC/SIC ratio and their concentration inhibition curves. Using the homologous series of sodium alkyl sulfates as a standard reference, we showed that PNPG was more effective than STS, which was the most effective of the homologous series. PEA was less effective than sodium decyl sulfate but more effective than sodium octyl sulfate. Motility tests in liquid medium and electron microscope investigations indicated that the modes of action of the three compounds, all of which effectively inhibit the swarming of Proteus, are different. Whereas STS and PEA inhibit swarming by inhibition of motility, PNPG seems to act on the swarming mechanism sensu strictori, without impairment of motility. STS immobilizes by inhibition of flagellum formation or by some lytic action on the flagella already synthesized. PEA acts by impairing flagellar function, but leaves the flagella morphologically intact.     

Inhibition of Swarming of Proteus by Sodium Tetradecyl Sulfate, beta-Phenethyl Alcohol, and p-Nitrophenylglycerine

The effects of sodium tetradecyl sulfate (STS), β-phenethyl alcohol (PEA), and p-nitrophenylglycerol (PNPG) on motility, swarming, flagellation, and growth of Proteus were examined. Growth-inhibitory concentrations (GIC) and swarming-inhibitory concentrations (SIC) were determined. A characterization of the swarming-inhibitory efficacy of these compounds was based on their GIC/SIC ratio and their concentration inhibition curves. Using the homologous series of sodium alkyl sulfates as a standard reference, we showed that PNPG was more effective than STS, which was the most effective of the homologous series. PEA was less effective than sodium decyl sulfate but more effective than sodium octyl sulfate. Motility tests in liquid medium and electron microscope investigations indicated that the modes of action of the three compounds, all of which effectively inhibit the swarming of Proteus, are different. Whereas STS and PEA inhibit swarming by inhibition of motility, PNPG seems to act on the swarming mechanism sensu strictori, without impairment of motility. STS immobilizes by inhibition of flagellum formation or by some lytic action on the flagella already synthesized. PEA acts by impairing flagellar function, but leaves the flagella morphologically intact.     

Immunohistochemical absence of adrenergic neurons in the dorsal part of the solitary tract nucleus in sudden infant death

The immunohistochemical distribution of TH and PNMT containing neuronal elements was investigated utilizing peroxidase anti-peroxidase methods in newborn control and sudden infant death syndrome (SIDS) brainstems. The TH immunoreactive neurons, within the medulla oblongata, displayed a similar distribution in both control and SIDS tissue. However, PNMT immunoreactive neurons seen in the dorsal part of the nucleus of tractus solitarius in control tissue were not observed in SIDS tissue. This alteration of adrenergic neurons in the dorsal part of NTS (region reported to be implicated in the control of blood pressure and respiration) could explain the cardiorespiratory disorders in SIDS.     

Agonist-induced desensitization of cholinergically stimulated phosphoinositide breakdown is independent of endogenously activated protein kinase C in HT-29 human colon carcinoma cells.

Activation of M3 muscarinic receptors in HT-29 cells by carbachol rapidly increases polyphosphoinositide breakdown. Pretreatment of these cells with carbachol (0.1 mM) for 5 h completely inhibits the subsequent ability of carbachol to increase [3H]inositol monophosphate ([3H]InsP) accumulation, paralleled by a total loss of muscarinic binding sites. In contrast, protein kinase C (PK-C)-mediated desensitization by incubation with phorbol esters [PMA (phorbol 12-myristate 13-acetate)], leading to a time- and dose-dependent inhibition of cholinergically stimulated InsP release (95% inhibition after 4 h with 0.1 microM-PMA), is accompanied by only a 40% decrease in muscarinic receptor binding, which suggests an additional mechanism of negative-feedback control. Neither carbachol nor PMA pretreatment had any effect on receptor affinity. Incubation with carbachol for 15 min caused a small increase of membrane-associated PK-C activity (15% increase, P less than 0.05) as compared with the potency of phorbol esters (PMA) (3-4-fold increase, P less than 0.01). Long-term incubation (4-24 h) with PMA resulted in a complete down-regulation of cytosolic and particulate PK-C activity. Stimulation of InsP release by NaF (20 mM) was not affected after a pretreatment with phorbol esters or carbachol, demonstrating an intact function of G-protein and phospholipase-C (PL-C) at the effector side. Determination of PL-C activity in a liposomal system with [3H]PtdInsP2 as substrate, showed no change in PL-C activity after carbachol (13 h) and short-term PMA (2.5 h) pretreatment, whereas long-term preincubation with phorbol esters (13 h) caused a small but significant decrease in PL-C activity (19%, P less than 0.05). Our results indicate that agonist-induced desensitization of phosphoinositide turnover occurs predominantly at the receptor level, with a rapid loss of muscarinic receptors. Exogenous activation of PK-C by phorbol esters seems to dissociate the interaction between receptor and G-protein/PL-C, without major effects on total cellular PL-C activity.     

Dosimetric mapping in BIORACK on IML2

Seven detector packages consisting of plastic nuclear track detectors, nuclear emulsions and thermoluminescence dosimeters were exposed in different locations inside BIORACK during the IML2 mission. The detectors supplement each other in their registration characteristics and cover well the different contributions of the space radiations to the dose. In this report, results are given on total dose measurements, cosmic ray flux and neutron dose. Total doses differ by up to a factor of 1.5 and heavy ion fluxes by more than a factor of 6 in the different locations. The results are compared with those of previous missions. The mission equivalent dose for the astronauts was calculated from the measurements to be 3.8 mSv.     

Proteolytic cleavage of bovine herpesvirus 1 (BHV-1) glycoprotein gB is not necessary for its function in BHV-1 or pseudorabies virus.

Glycoprotein B homologs represent the most highly conserved group of herpesvirus glycoproteins. They exist in oligomeric forms based on a dimeric structure. Despite the high degree of sequence and structural conservation, differences in posttranslational processing are observed. Whereas gB of herpes simplex virus is not proteolytically processed after oligomerization, most other gB homologs are cleaved by a cellular protease into subunits that remain linked via disulfide bonds. Proteolytic cleavage is common for activation of viral fusion proteins, and it has been shown that herpesvirus gB homologs are essential for membrane fusion events during infection, e.g., virus penetration and direct viral cell-to-cell spread. To analyze the importance of proteolytic cleavage for the function of gB homologs, we isolated a mutant bovine herpesvirus 1 (BHV-1) expressing a BHV-1 gB that is no longer proteolytically processed because of a deletion of the proteolytic cleavage site and analyzed its phenotype in cell culture. We showed previously that BHV-1 gB can functionally substitute for the homologous glycoprotein in pseudorabies virus (PrV), based on the isolation of a PrV gB-negative PrV recombinant that expresses BHV-1 gB (A. Kopp and T. C. Mettenleiter, J. Virol, 66:2754-2762, 1992). Therefore, we also isolated a mutant PrV lacking PrV gB but expressing a noncleavable BHV-1 gB. Our results show that cleavage of BHV-1 gB is not essential for its function in either a BHV-1 or a PrV background. Compared with the PrV recombinant expressing cleavable BHV-1 gB, deletion of the cleavage site in the recombinant PrV did not detectably alter the viral phenotype, as analyzed by plaque assays, one-step growth kinetics, and penetration kinetics. In the BHV-1 mutant, the uncleaved BHV-1 gB was functionally equivalent to the wild-type protein with regard to penetration and showed only slightly delayed one-step growth kinetics compared with parental wild-type BHV-1. However, the resulting plaques were significantly smaller, indicating a role for proteolytic cleavage of BHV-1 gB in cell-to-cell spread of BHV-1.