Early association of reactive astrocytes with senile plaques in Alzheimer's disease

The fibrillar beta-amyloid protein (A beta) plaques of Alzheimer's disease (AD) are associated with reactive astrocytes and dystrophic neurites and have been suggested to contribute to neurodegenerative events in the disease. We recently reported parallel in vitro and in situ findings, suggesting that the adoption of a reactive phenotype and the colocalization of astrocytes with plaques in AD may be mediated in large part by aggregated A beta. Thus, A beta-mediated effects on astrocytes may directly affect disease progression by modifying the degenerative plaque environment. Alternatively, plaque-associated reactive astrocytosis may primarily represent a glial response to the neural injury associated with plaques and not significantly contribute to AD pathology. To investigate the validity of these two positions, we examined the differential colocalization of reactive astrocytes and dystrophic neurites with plaques. Hippocampal sections from AD brains--ranging in neuropathology from mild to severe--were triple-labeled with antibodies recognizing A beta protein, reactive astrocytes, and dystrophic neurites. We observed not only plaques containing both or neither cell type, but also plaques containing (1) reactive astrocytes but not dystrophic neurites and (2) dystrophic neurites but not reactive astrocytes. The relative proportion of plaques colocalized with reactive astrocytes in the absence of dystrophic neurites is relatively high in mild AD but significantly decreases over the course of the disease, suggesting that plaque-associated astrocytosis may be an early and perhaps contributory event in AD pathology rather than merely a response to neuronal injury. These data underscore the potentially significant contributions of reactive astrocytosis in modifying the plaque environment in particular and disease progression in general.     

Identification of the substrate and pseudosubstrate binding sites of phosphorylase kinase gamma-subunit

Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase gamma-subunit (Huang, C.-Y.F., Yuan C.-J., Livanova, N.B., and Graves, D.J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) and noncompetitive inhibition versus ATP. PhK5 showed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154).     

Novel interneuron having hybrid modulatory-central pattern generator properties in the feeding system of the snail, Lymnaea stagnalis

1. We used intracellular recording techniques to examine the role of a novel type of protraction phase interneuron, the lateral N1 (N1L) in the feeding system of the snail Lymnaea stagnalis. 2. The N1Ls are a bilaterally symmetrical pair of electrotonically coupled interneurons located in the buccal ganglia. Each N1L sends a single axon to the contralateral buccal ganglia. Their neurite processes are confined to the buccal neuropile. 3. In the isolated CNS, depolarization of an N1L is capable of driving a full (N1-->N2-->N3), fast (1 cycle every 5 s) fictive feeding rhythm. This was unlike the previously described N1 medial (N1M) central pattern generator (CPG) interneurons that were only capable of driving a slow, irregular rhythm. Attempts to control the frequency of the fictive feeding rhythm by injecting varying amounts of steady current into the N1Ls were unsuccessful. This contrasts with a modulatory neuron, the slow oscillator (SO), that has very similar firing patterns to the N1Ls, but where the frequency of the rhythm depends on the level of injected current. 4. The N1Ls' ability to drive a fictive feeding rhythm in the isolated preparation was due to their strong, monosynaptic excitatory chemical connection with the N1M CPG interneurons. Bursts of spikes in the N1Ls generated summating excitatory postsynaptic potentials (EPSPs) in the N1Ms to drive them to firing. The SO excited the N1M cells in a similar way, but the EPSPs are strongly facilitatory, unlike the N1L-->N1M connection. 5. Fast (1 cycle every 5 s) fictive feeding rhythms driven by the N1L occurred in the absence of spike activity in the SO modulatory neuron. In contrast, the N1L was usually active in SO-driven rhythms. 6. The ability of the SO to drive the N1L was due to strong electrotonic coupling, SO-->N1L. The weaker coupling in the opposite direction, N1L-->SO, did not allow the N1L to drive the SO. 7. Experiments on semintact lip-brain preparations allowed fictive feeding to be evoked by application of 0.1 M sucrose to the lips (mimicking the normal sensory input) rather than by injection of depolarizing current. Rhythmic bursting, characteristic of fictive feeding, began in both the SO and N1L at exactly the same time, indicating that these two cell types are activated in "parallel" to drive the feeding rhythm. 8. The N1L is also part of the CPG network. It Excited the N2s and inhibited the N3 phasic (N3p) and N3 tonic (N3t) CPG interneurons like the N1Ms.(ABSTRACT TRUNCATED AT 400 WORDS)     

Respiratory pentane: a measure of in vivo lipid peroxidation applied to rats fed diets varying in polyunsaturated fats, vitamin E, and selenium and exposed to nitrogen dioxide

Pentane is one decomposition product of omega 6-unsaturated lipid hydroperoxides. The measurement of respiratory pentane is one of the most sensitive in vivo tests of lipid peroxidation. This measurement was applied to test the ability of a low level, short-term exposure of rats of nitrogen dioxide to induce lipid peroxidation. When exposed to 4.48 ppm nitrogen dioxide for 60 min, no increase in pentane production was detected. For 10 weeks prior to exposure, the rats were fed Torula yeast-based diets with 10% stripped lard or 10% stripped corn oil. The ratios of basal pentane production by rats fed the following antioxidants were for corn oil- and lard-fed rats, respectively: 40 I.U. vitamin E/kg and 0 selenium: 0 vitamin E and 0.1 ppm selenium: 0 vitamin E and 0 selenium, 1:2.2:5.8 and 1:1.9:3.5. Pentane production was significantly (P<0.05) greater by corn oil-fed rats than by lard-fed rats only when both vitamin E and selenium were absent from the diet.     

Position and organization of thalamic cellular activity during diencephalic recording. I. Pressure-evoked activity

With the aid of a computer graphic display technique, data gathered from exploratory diencephalic recordings in 130 stereotactic operative procedures are presented to show the location and somatotopography of 'pressure'-evoked cellular responses. In addition, the variable position of these responses in relation to common diencephalic landmarks is presented. Our findings indicate that response points are more compact when related to a common posterior commissure (PC) and lie in a sickle-shaped zone 14-16 mm lateral to the midline and 2-8 mm rostral to anatomically the nucleus ventralis caudalis or the nucleus ventralis intermedius. Somatotopographically, face responses were medial to the leg.     

The effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro

Effects of dexamethasone and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were studied in cultures of adult human marrow stromal cells. In primary culture, dexamethasone (10(-8) M) increased the number of fibroblast colonies formed but decreased their average size. The number of colonies expressing alkaline phosphatase activity was increased, consistent with the enhancement of osteogenic differentiation by this glucocorticoid. In secondary culture, osteogenic differentiation was assessed by measurement of the steady-state levels of particular mRNAs that are characteristic of cells of the osteoblast lineage. The mRNAs for alpha 1(I)-procollagen, alkaline phosphatase, osteopontin and bone sialoprotein were expressed under all culture conditions used. In contrast, osteocalcin mRNA expression was detectable only in cultures treated with 1,25(OH)2D3 (10(-8) M). Addition of 1,25(OH)2D3 to control increased the expression of the mRNAs for alkaline phosphatase and osteopontin but had no significant effect on bone sialoprotein expression. The highest levels of expression of the mRNAs for alkaline phosphatase, bone sialoprotein and osteocalcin were observed in dexamethasone-treated cultures to which 1,25(OH)2D3 had been added. These results demonstrate that, as earlier found in other species, dexamethasone and 1,25(OH)2D3 promote the osteogenic differentiation of human marrow stromal cells as measured by expression of these osteogenic markers.     

Fracture of the radial forearm osteocutaneous donor site

Since the introduction of the osteocutaneous radial forearm flap in 1983, fractures of the radius have been reported to occur in approximately 30% of cases. Fracture of the donor forearm has been the cause of the most significant morbidity, and the difficulty in management of these fractures has been reported. We report our experience in managing three fractures involving the donor forearm. Optimum results can be achieved by early stabilization with external fixation and vascularized bone grafting. Excessive resection of the radius should be avoided and alternative sources of vascularized bone used to avoid mutilation of the forearm.     

Lack of effect of hematopoietic growth factors on human breast epithelial cell growth in serum-free primary culture

A number of recombinant cytokines believed to regulate normal hematopoiesis are now being used in cancer treatment protocols to reduce the myelosuppressive toxicity of intensive chemoradiotherapy regimens. It is widely assumed that such cytokines are relatively specific for hematopoietic cells, although some cell lines derived from a variety of non-hematopoietic human tumors can respond to some of these factors. However, relatively little is known about their ability to stimulate (or inhibit) the proliferation of freshly isolated normal or malignant non-hematopoietic cells. We have used a serum-free culture medium that selectively supports the growth of human breast epithelial cells (HBEC) obtained directly from normal or malignant tissue samples to evaluate potential stimulatory or inhibitory effects of eight cytokines: granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Steel factor, interleukin-2, interleukin-3, interleukin-6, transforming growth factor-beta and macrophage inflammatory protein-1 alpha, on these cells cultured both in the presence of epidermal growth factor, a potent stimulator of HBEC growth, and in its absence. HBEC growth was assessed after 7 and 14 days using the tetrazolium-dye reduction assay. Potential effects on the well studied MCF-7 breast cancer cell line, cultured under the same conditions, were also investigated. None of the cytokines (which were tested over a wide range of concentrations) had any modulating effect on the growth of normal or malignant HBEC under the conditions used with the exception of transforming growth factor-beta, which was consistently and significantly inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased cortisol response to exogenous adrenocorticotropic hormone in chronically stressed pigs: influence of housing conditions

In a longitudinal experiment, the influence of tethered housing (a condition of chronic stress) on the reactivity of the adrenal cortex to exogenous ACTH was investigated in gilts. To that end, the plasma cortisol response to synthetic ACTH (1-24; 10 micrograms/kg of BW; i.v. bolus injection via a permanent catheter) was determined before and after prolonged tethered housing. Two systems for tethered housing were used, one more restrictive than the other with regard to possibilities for visual and tactile contacts with conspecifics and visual control over the environment. The ACTH treatment induced a marked, transient plasma cortisol response in all gilts studied, irrespective of their housing conditions. Long-term tethered housing increased the ACTH-induced cortisol response. Possible effects of the experimental procedure or age-related effects could be excluded, because in control gilts, which were housed loose during the entire experimental period, the cortisol response to ACTH remained unaltered. The chronic stress-induced increase in the ACTH-induced cortisol response was considerably more pronounced and persistent in gilts that were deprived of possibilities for social contacts with conspecifics and visual control over the environment than in gilts with such possibilities. These data indicate that in tethered gilts adaptational changes occur at the level of the adrenal cortex that affect the ACTH-induced adrenocortical response. In addition, not only physical restraint but also restriction of social contact and visual control play an important role in the development of these changes.