Anterograde Axonal Transport of Peptidylglycine α-Amidating Monooxygenase in Rat Sciatic Nerves

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.     

Protein kinase A inhibitors enhance radiation-induced apoptosis

In addition to a role for de novo protein synthesis in apoptosis we have previously shown that activation of a protein phosphatase or loss of activity of a kinase is also important in radiation-induced apoptosis in human cells [Baxter, and Lavin (1992): J Immunol 148:149–1954]. We show here that some inhibitors of protein kinases exacerbate radiation-induced apoptosis in the human cell line BM13674. The specific protein kinase A inhibitor isoquinoline sulfonamide (20 μM) gave rise to significantly increased levels of apoptosis at 2–6 h postirradiation compared to values after radiation exposure only. The same concentration of isoquinolinesulfonamide, which was effective in increasing apoptosis, reduced activity markedly. A 66% inhibition of cyclic AMP-dependent protein kinase A activity occurred in unirradiated cells at this concentration of H89 and activity was reduced to 58% in irradiated cells. Calphostin C, a specific inhibitor of protein kinase C, at a concentration of 0.1 μM, which caused 68% inhibition of enzyme activity in irradiated cells, failed to enhance the level of radiation-induced apoptosis. Other kinase inhibitors did not lead to an additional increase in apoptosis over and above that observed after irradiation. The results obtained here provide further support for an important role for modification of existing proteins during radiation-induced apoptosis.     

Dissipative particle dynamics simulation of a gold nanoparticle system

Dissipative particle dynamics (DPD) was carried out to study systems containing gold atoms, organic ether (oligohydroquinonyl ether terminated with a thiol group) and organic solvents. The components in the simulated system are very different in size and chemical nature. Our simulation showed that the reproduction of the macroscopic experimental phase separation, properly dividing the polymeric molecule into beads, selecting the size of gold bead, and choosing the appropriate interaction parameters between beads are crucial. In addition, the solvent effect was the dominant factor for the formation of spherical aggregates of Au atoms and organic ether molecules. We report the interaction strengths between the solvent and gold clusters. Our work has demonstrated that DPD methods can be applied to the study of complex meso-scale systems.     

Nutrient and chemical sensing by intestinal pathogens

Pathogenic gut bacteria, such as those comprising the Enterobacteriaceae family, have evolved sophisticated virulence mechanisms, including nutrient and chemical sensing, to escape host defense strategies and produce disease. In this review we describe the mechanisms utilized by the enteric pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 to achieve successful colonization of its mammalian host.     

Fractal root system of Melica przewalskyi along different aspect in degraded grassland

Aims Fractal root system is phenotypic plasticity result of plant root architecture to respond to environmental heterogeneity, may reflect the growth strategy of plants to adapt to environmental conditions. Our objective was to explore the relationship between root fractal dimension and fractal abundance of fractal root system of Melica przewalskyi population in response to aspect variation in the northwest of China. Methods The study site was located in a degraded alpine grassland on the northern slope in Qilian Mountains, Gansu Province, China. Survey and sampling were carried out at 40 plots which were set up along four slope aspects transects with 20 m distance between adjacent plots. Handheld GPS was used to determine the elevation, longitude and latitude of each plot. ArcGIS was used to set up digital elevation model (DEM). Community traits were investigated and six individuals roots of M. przewalskyi were collected randomly at each plot. The samples were cleaned and divided into different organs, then scanning the root with the Win-RHIZO for measurements of fractal dimension and fractal abundance in laboratory, and their biomass were then measured after being dried at 80 °C in an oven. Important findings With the slope aspect turned from north to east, west, and south, the density, height and soil moisture content of the plant community displayed a pattern of initial decline, the height, density, root fractal abundance of M. przewalskyi increased and the root fractal dimension decreased. The root fractal dimension was negatively associated with the fractal abundance in all aspects, but the relationship varied along the slope aspects gradient; there was a highly significant negative correlation (p < 0.01) between the root fractal dimension and fractal abundance at north slope and south slope aspect, whereas the correlation only reached a significant level (p < 0.05) at the east slope aspect and west slope aspect; indicating that there is a trade-off between the root fractal dimension and fractal abundance. In addition, when the slope aspect changed from north to east, west and south, the standardized major axis (SMA) slope of the regression equation in the scaling relationships between root fractal dimension and fractal abundance increased (p < 0.05), indicating that the roots of M. przewalskyi at the droughty southern slope have less branch and more sparse in the same soil volume of root exploitation and utilization. Consequently, the resource allocation pattern on reasonable trade-off between root fractal dimension and fractal abundance in different slope aspect of M. przewalskyi, reflects the relationship between the income and the cost of construction of plant root architecture.     

Inhibitory effect of aspirin on cholera toxin-induced phospholipase and cyclo-oxygenase activity

Cholera toxin (CT) stimulated phospholipase activity and caused [3H]arachidonic acid (3H-AA) release in a murine macrophage/monocyte cell line. Pretreatment of cells with dexamethasone, a phospholipase A2 (PLA2) inhibitor, did not affect CT-induced 3H-AA release. In contrast, aspirin, which is an inhibitor of phospholipase C (PLC), blocked CT-induced 3H-AA release and subsequent prostaglandin (PC) synthesis. The inhibitory effect of aspirin was dose dependent, with 4 mM reducing the CT response by approximately 50%. Similarly, inhibition was time dependent, occurring when the drug was added to the culture medium as late as 30 min after CT. Brief exposure (30 min) of the cells to aspirin did not alter their subsequent response to CT, but 3H-AA release from cells exposed to aspirin for 2.5 h was irreversibly inhibited. The data suggested that CT stimulation of AA metabolism may involve increased PLC activity.     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

Development of a one-step enzyme immunoassay for the quantitation of gibberellin A3 in barley seeds

The development of a sensitive and specific enzyme immunoassay for GA₃ is reported. This method was based on the use of peroxidase labelled GA₃ and immobilized antibodies. In order to obtain a rapid immunoassay, several steps of purification were analyzed to show their necessity. Barley seed extracts were assayed at different steps of purification to exhibit the effect of extract components on the assay. It was demonstrated that HPLC had to be performed when a selective quantitation of GA₃ was required. This assay allowed GA₃ to be measured with reproducibility as its unmethylated form and the quantitation of GA₃ in barley seeds with this enzyme immunoassay was correlated to a GC-MS method.Abbreviations GA₃ gibberellin A₃ - EIA enzyme immunoassay - DMF dimethylformamide - TEA tri(n)ethylamine - BSA bovine serum albumin - OVA ovalbumine - ECF ethylchloroformate - PB phosphate buffer     

Genic homozygosity in an ancient reptile (Alligator mississippiensis)

Proteins assayed electrophoretically showed variation at only three of 49 presumed genetic loci in alligators from southwestern Louisiana. Average heterozygosity per individual was 0.021±0.012; proportion of polymorphic loci was 0.06. Data on the history, structure, and ecology of this alligator population are consistent with natural selection as the primary factor accounting for this low genetic variability. However, neither a historic population bottleneck nor some genetic mechanism limiting variability can be dismissed as a possible factor.The study was supported by NSF Grant BMS 73-0125 to H.C.D.