Phorbol Myristate Acetate and 8-Bromo-Cyclic AMP-Induced Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin in Astrocytes: Comparison of Phosphorylation Sites

Both the protein kinase C (PK-C) activator, phorbol 12-myristate 13-acetate (PMA), and the cyclic AMP-dependent protein kinase (PK-A) activator, 8-bromo-cyclic AMP (8-BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8-BR results in the morphological transformation of flat, polygonal-shaped cells into stellate, process-bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8-BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8-BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8-BR treatment was restricted to serine residues in the N-terminal domain of these proteins. Studies were also conducted to compare the two-dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8-BR with those produced when the proteins were phosphorylated with purified PK-C or PK-A. PK-C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK-C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative Microdialysis: Analysis of Transients and Application to Pharmacokinetics in Brain

The behavior of a microdialysis probe in vivo is mathematically described. A diffusion-reaction model is developed that not only accounts for transport of substances through tissues and probe membranes but also accounts for transport across the microvasculature and metabolism. Time-dependent equations are presented both for the effluent microdialysate concentration and for concentration profiles about the probe. The analysis applies either to measuring the tissue pharmacokinetics of drugs administered systemically, or for sampling of endogenously produced substances from tissue. In addition, an expression is developed for the transient concentration about the probe when it is used as an infusion device. All mathematical expressions are found to be a sum of an algebraic and an integral term. Theoretical prediction of time-dependent probe behavior in brain has been compared with experimental data for acetaminophen administered at 15 mg/kg to rats by intravenous bolus. Plasma and whole striatal tissue samples were used to describe plasma kinetics and to estimate a capillary permeability-area product of 0.07 min-1. Theoretical prediction of transient effluent dialysate concentrations exhibited close agreement with experimental data over 60 min. Terminal decline of the dialysate effluent concentration was slightly overestimated but theoretical concentrations still lay within the 95% confidence interval of the experimental data at 112 min. Microvasculature transport and metabolism play major roles in determining microdialysate transient responses. Extraction fraction (recovery) has been shown to be a declining function in time for five probe operating conditions. High rates of metabolism and/or capillary transport affect the time required to approach steady-state extraction, shortening the time as the rates increase. Conversely, for substances characterized by low permeabilities and negligible metabolism, experimental situations exist that are predicted to have very slow approaches to microdialysis steady state.     

Comparison of Representative Strains of Infectious Hematopoietic Necrosis Virus by Serological Neutralization and Cross-Protection Assays

Infectious hematopoietic necrosis virus (IHNV) is a pathogen of young salmon and trout. Viral epizootics among these fish in private and public rearing facilities have been a problem in the northwestern United States from California to Alaska, and an IHNV vaccine has been sought by the aquaculture experts. Since an IHNV vaccine must be designed to immunize against all viral serotypes, an analysis of IHNV serotypes was made. A large number of viruses from widely separated geographic locations and different fish species had already been placed in one of five electropherotypes by the migration of the virion proteins in sodium dodecyl sulfate-polyacrylamide gels. Also, there was evidence that some of these virus isolates had differences in virulence for chinook salmon, rainbow trout, or kokanee salmon. Previous serological studies with polyclonal rabbit antisera and three IHNV isolates indicated that there was only one serotype (B. B. McCain, J. L. Fryer, and K. S. Pilcher, Proc. Soc. Exp. Biol. Med. 137:1042-1046, 1971). A substantial number of new IHNV isolations have been made since that study, and thus a more extensive comparison was made of 10 different IHNV isolates representing the five electropherotypes. This report shows that the glycoprotein from a single isolate of IHNV can induce a protective immune response in vivo to the five IHNV electropherotypes. Plaque reduction neutralization assays indicated that there was only one serotype. Thus, despite the differences observed in the migration of the structural proteins for IHNV isolated from separate geographic locations and different fish species, only one neutralizing virus type was identified.     

Long-term effects of dexamethasone and nerve growth factor on adrenal medullary cells cultured from young adult rats

Summary Normal postnatal rat chromaffin cells and rat pheochromocytoma cells are known to show extensive Nerve Growth Factor (NGF)-induced process outgrowth in culture, and this outgrowth from the postnatal chromaffin cells is abolished by the corticosteroid dexamethasone. To determine whether adult rat chromaffin cells respond to NGF and dexamethasone, dissociated adrenal medullary cells from 3-month-old rats were cultured for 30 days in the presence or absence of these agents. Such cultures contained typical chromaffin cells, chromaffin cells with processes, and neurons. Fewer than 2 % of normal adult chromaffin cells formed processes under any of the conditions studied, and statistically significant changes in this proportion were not detectable in the presence of NGF or dexamethasone. Adrenal medullary neurons, however, were observed only in the presence of NGF, in cultures with or without dexamethasone, and thus appear to be previously unreported NGF targets which require NGF for survival or process outgrowth. Dexamethasone markedly increased total catecholamine content, total content of epinephrine, and tyrosine hydroxylase activity in cultures with or without NGF. In contrast, postnatal rat chromaffin and rat pheochromocytoma cells which have been studied in culture do not produce epinephrine under any of these conditions. It is concluded that rat adrenal chromaffin cells undergo age-related changes in both structural and functional plasticity. The in vitro characteristics of rat pheochromocytoma cells more closely resemble those of postnatal than of adult rat chromaffin cells, but may not entirely reflect the properties of the majority of chromaffin cells in either age group.     

Terbium ion binding to Limuluspolyphemus hemocyanin

Terbium ion binds to calcium-free $\text{Limulus}$ hemocyanin at pH 7.0 and 8.9, and promotes the aggregation of hemocyanin subunits, a phenomenon associated with calcium binding. An excitation maximum for the bound terbium at 293 nm and the results of treating the hemocyanin with N-bromosuccinimide indicate that energy transfer from tryptophan to the bound terbium is responsible for the enhancement of terbium fluorescence. At pH 8.9, addition of calcium to hemocyanin containing bound terbium results in only a partial loss of terbium fluorescence, suggesting heterogeneity in the terbium binding sites. Titration of hemocyanin with terbium also indicates multiple binding sites.     

The effects of ambient flow velocity,colony size,and upstream colonies on the feeding success of bryozoa. I. Bugula stolonifera Ryland,an arborescent species

The effects of ambient flow velocity, colony size, and the presence of upstream colonies on the feeding success of the arborescent bryozoan, Bugula stolonifera (Ryland), were studied. Faster ambient flow velocities were found to reduce feeding of zooids of small colonies but not of large colonies. Zooids from different regions of colonies dominated in feeding at different ambient flow velocities: upstream zooids dominated in feeding from slow ambient flow: zooids from central regions dominated in feeding from fast ambient flow. These results are interpreted with respect to the branching morphology of colonies. Finally, evidence that upstream colonies interfere with the feeding success of zooids of colonies downstream was obtained.     

Benthic macrofaunal communities in intermittent estuaries during a drought: Comparisons with permanently open estuaries

Drought conditions have prevailed in many areas of NSW since 2002. On the mid-north coast, below-average rainfall resulted in reduced riverine flows and the extended closure of intermittent estuaries within the Solitary Islands Marine Park. Patterns of structure of benthic infaunal communities were evaluated at the height of the drought to determine if they differed between closed, intermittent estuaries and permanently open estuaries within the region. Replicate van Veen grab samples were taken in the upper, mid- and lower reaches of six intermittent and three permanently open estuaries and sieved to retain the macrofauna. A range of physico-chemical measures was also taken at each sampling time. Multivariate analyses of assemblage data revealed a significant difference between the structure of the two estuary types and also among estuaries within each type. Differences between estuary types were attributable to small differences in the abundance of a number of taxa but also to the absence of the amphipod Urohaustorius metungi from most of the intermittent estuaries. In contrast, these small amphipods dominated communities in the lower reaches of the permanently open estuaries. Physico-chemical variables were highly variable among estuaries and were not strongly correlated with assemblage patterns. Correlations with catchment size were the strongest and, as most of the intermittent estuaries in the region are smaller than the permanently open estuaries, this confounds the interpretation of assemblage patterns in this preliminary study. In order to differentiate between the effects of catchment size and entrance status, the same estuaries need to be resurveyed during periods when at least some of the intermittent estuaries are open.