Effect of age on bone mineral density and micro architecture in the radius and tibia of horses: an Xtreme computed tomographic study.

BACKGROUND: The effect of age on the bone mineral density and microarchitecture of the equine radius and tibia was investigated. Fifty-six bones from 15 horses aged four to 21 years were used. There were nine geldings and six mares, and none of the horses had any disease influencing bone properties. Xtreme computed tomography was used to evaluate a 9-mm segment of the diaphysis and metaphysis of each bone. The following variables were determined: length of the bone, circumference and diameter in the frontal and sagittal planes in the middle of the bone.Diaphysis: total volume, bone volume, bone volume ratio, slice area, bone area, marrow area, cortical and marrow thickness, bone mineral density, polar moment of inertia of the cortex.Metaphysis: total area, bone area, cortical bone area, cortical thickness, bone mineral density, bone mineral density in the cortex, bone mineral density in the trabecular region, trabecular number, trabecular thickness, trabecular separation, polar moment of inertia of the metaphysis, polar moment of inertia of the cortex of the metaphysis. RESULTS: Bone density and microarchitecture were not affected by breed or gender. However, the microarchitecture varied with the age of the horse; the number of trabeculae decreased significantly and the distance between trabeculae increased significantly with increasing age. There were no significant differences between bones of the left and right limbs or between the radius and tibia. CONCLUSION: The variables investigated did not differ between geldings and mares. However, there were age-related changes in the microstructure of the bones. Further experimental studies are necessary to determine whether these changes reduce bone strength. Age-related changes in the bones were seen and may explain the higher incidence of fractures and fissures in older horses.

Repacking of the transmembrane domains of P-glycoprotein during the transport ATPase cycle.

P-glycoprotein (P-gp) is an ABC (ATP-binding cassette) transporter, which hydrolyses ATP and extrudes cytotoxic drugs from mammalian cells. P-gp consists of two transmembrane domains (TMDs) that span the membrane multiple times, and two cytoplasmic nucleotide-binding domains (NBDs). We have determined projection structures of P-gp trapped at different steps of the transport cycle and correlated these structures with function. In the absence of nucleotide, an approximately 10 A resolution structure was determined by electron cryo-microscopy of two-dimensional crystals. The TMDs form a chamber within the membrane that appears to be open to the extracellular milieu, and may also be accessible from the lipid phase at the interfaces between the two TMDs. Nucleotide binding causes a repacking of the TMDs and reduction in drug binding affinity. Thus, ATP binding, not hydrolysis, drives the major conformational change associated with solute translocation. A third distinct conformation of the protein was observed in the post-hydrolytic transition state prior to release of ADP/P(i). Biochemical data suggest that these rearrangements may involve rotation of transmembrane alpha-helices. A mechanism for transport is suggested.

Transport of L-citrulline in neural cell cultures.

Uptake of L-[(14)C]citrulline was studied in cell culture models of the main neural cell populations, in astroglia-rich primary cultures derived from neonatal rat brain, in rat glioma cells C6-BU-1, in cells of the murine microglial clone N11 and in the glioma x neuroblastoma hybrid cell line 108CC15 with neuronal properties. For comparison, cells of the peripheral macrophage cell line RAW 264.7 were also investigated. A saturable component of uptake was found in all cases with K(M) values between 0.4 and 3.4 mM and V(max) values between 15 and 35 nmol.min(-1).(mg protein)(-1). A nonsaturable component dominated uptake at high concentrations of extracellular citrulline. Rates of uptake of L-citrulline were not affected when Na(+) or Cl(-) were omitted from the incubation medium or in the presence of depolarizing concentrations of K(+). Saturable uptake of citrulline was strongly inhibited by an excess of histidine or beta-2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid; excess amounts of arginine, creatine, glutamate, cysteic acid or N-methyl-alpha-aminoisobutyric acid did not reduce citrulline uptake. Preincubation of the cells with bacterial lipopolysaccharide and interferon gamma did not stimulate transport of citrulline. The results suggest that at physiological concentrations citrulline is taken up by neural cells with the help of transport system L for large neutral amino acids. Therefore, in the brain, effective utilization of extracellular citrulline as part of an intercellular trafficking of intermediates of an NO/citrulline cycle depends on the concentrations of all neutral amino acids present.

Induction of argininosuccinate synthetase in rat brain glial cells after striatal microinjection of immunostimulants.

The enzyme argininosuccinate synthetase (ASS) initiates the metabolic pathway leading from L-citrulline to L-arginine, the only physiological substrate of all isoforms of nitric oxide synthases. The presence of ASS in glial cells in vivo was investigated by immunohistochemical methods in a model of rat brain inflammation. Phosphate-buffered saline or a mixture of bacterial lipopolysaccharide and interferon-gamma was injected into the left striatum, and animals were killed 24 hours later. Ipsilateral and contralateral sides of brain sections were incubated with an antiserum against ASS or antibodies against cell-specific markers. In the three areas examined, striatum, corpus callosum, and cortex, a strong induction of ASS immunoreactivity was observed in glial cells after injection of immunostimulants. A detailed quantitative analysis of double-stained sections revealed that ASS was almost exclusively expressed in reactive, ED1-positive microglial cells/brain macrophages in immunostimulant- or sham-injected ipsilateral sides of the sections. Furthermore, ASS/ED1 costaining was observed in perivascular cells. Colocalization of ASS with astroglial marker glial fibrillary acidic protein was given only occasionally after immunostimulation. ASS-positive neurons were detected in control and experimental animals; staining intensity was comparable in both cases. The results suggest that neurons express ASS constitutively, whereas the enzyme is induced in glial cells in response to proinflammatory stimuli. This finding is the first demonstration of an induction of a pathway auxiliary to generation of nitric oxide in brain in response to immunostimulants and provides new insight into neural arginine metabolism.

Argininosuccinate synthetase: localization in astrocytes and role in the production of glial nitric oxide.

An antiserum raised against the peptide representing the partial sequence 196-222 of mouse liver argininosuccinate synthetase (ASS) was used to detect and localize the enzyme in cells of neural primary cultures. No ASS immunoreactivity was detected by Western blotting in homogenates of mouse pure astroglial cultures and rat astroglia-rich cultures. However, when the cultures had been treated with bacterial lipopolysaccharide, interferon-gamma, or a combination of both, ASS immunoreactivity was disclosed. Immunocytochemical examination of rat astroglia-rich cultures revealed a colocalization of ASS with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were some GFAP-positive cells showing no specific staining for ASS, and vice versa. Colocalization of ASS with the inducible isoform of nitric oxide synthase in the same cell was shown only occasionally; nitric oxide synthase was predominantly expressed in microglial cells. In rat neuron-rich primary cultures astroglial cells as well as neurons expressed ASS. Cells of mouse pure astroglial cultures were able to synthesize arginine and, consequently, nitric oxide from citrulline, but not from ornithine. The findings demonstrate that ASS is expressed in astroglial cells under conditions that stimulate long-lasting production of nitric oxide; a functional role of this enzyme in the latter process is implicated.

Presence of argininosuccinate synthetase in glial cells as revealed by peptide-specific antisera.

The presence and the possibility of induction of argininosuccinate synthetase in a glial cell line were investigated. For this purpose, antisera were produced against peptides representing partial sequences 196-222 and 337-349, respectively, of the mouse liver enzyme. Both antisera were shown to be monospecific for argininosuccinate synthetase. In Western blot experiments, immunoreactivity was found in mouse liver and brain homogenates. Only weak immunoreactivity was detectable in homogenates of cultured glioma cells, C6-BU-1. However, when the glioma cells were treated with either bacterial lipopolysaccharide, interferon-gamma, or a combination of both, argininosuccinate synthetase immunoreactivity was increased. The findings demonstrate that this enzyme is present in glial cells and is induced under conditions which stimulate persistent production of nitric oxide. The antisera will be a valuable tool for further investigations on arginine synthesis in brain as well as peripheral cells.

Stimulation of arginine transport and nitric oxide production by lipopolysaccharide is mediated by different signaling pathways in astrocytes.

Transport of L-arginine and generation of nitrite in microglia-free astroglial cultures derived from neonatal mouse brain were stimulated by bacterial lipopolysaccharide (LPS) in a time- and dose-dependent manner. LPS stimulated arginine transport between 1.3- and 2.5-fold; half-maximal stimulation was obtained with 0.3 micrograms/ml LPS. Acceleration of transport was detectable within 6 h of incubation with LPS. Cycloheximide or actinomycin D neutralized the effect of LPS. Stimulation of generation of nitrite was reduced when the cells were incubated simultaneously with LPS and either genistein or diethyldithiocarbamate, inhibitors of protein tyrosine kinase and nuclear transcription factor kappa, respectively. However, stimulation of arginine transport was not reduced in the presence of these compounds. Dexamethasone inhibited stimulation of nitric oxide (NO) production but not of arginine transport. Protein kinase C inhibitor staurosporine had no effect on either process. The results suggest that LPS-stimulated acceleration of arginine transport in astrocytes requires protein as well as RNA synthesis. Induction of synthesis of an astroglial cationic amino acid transport system appears to be mechanistically independent from stimulation of intracellular NO production.

Transport of L-arginine in cultured glial cells.

Uptake of radiolabelled L-arginine was studied in four different kinds of glial cultures, in astroglia-rich primary cultures derived from neonatal rat and mouse brains, in pure murine astrocyte cultures, and in rat glioma cells C6-BU-1. A saturable component of uptake was found in all cases with KM values between 15 and 35 microM and Vmax values between 0.8 and 2.5 nmol.min-1.(mg protein)-1. In addition, in all cell types a non-saturable component dominated total uptake at high concentrations of extracellular arginine. Rates of uptake of arginine were not affected when Na+ or Cl- were absent from the incubation buffer. Carrier-mediated uptake of arginine was reduced by depolarizing concentrations of K+ and strongly inhibited by an excess of lysine or ornithine. Histidine, asparagine, glutamine, citrulline, creatine, NG-nitro-L-arginine, NG-monomethyl-L-arginine, or L-canavanine inhibited L-arginine transport to various degrees. Uptake of arginine was not reduced in the presence of serine or alanine cysteic acid, N-methyl-alpha-aminoisobutyric acid, or 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. Rates of uptake of arginine were increased when cells had been preloaded with lysine. Preincubation of primary cultures, but not glioma cells, with bacterial lipopolysaccharide stimulated transport of arginine by increasing the Vmax value of uptake. This stimulation was dependent on protein synthesis. The results suggest that, at physiological concentrations, arginine is taken up into the glial cells with the help of the transport system "y+" for basic amino acids. In glial primary cultures, uptake of arginine appears to be regulated by compounds which also exert influence on nitric oxide synthesis.

Gender patterns in social touch: the impact of setting and age.

This study examined the impact of age of participants and the setting in which touch occurred on gender patterns in 799 instances of observed intentional touch. Gender asymmetry, a pattern in which men are more likely to touch women than vice versa, was observed when touch between adults was examined but not when touch involving children was examined. Adult touch interactions occurring in public, nonintimate settings showed gender asymmetry, but adult touch interactions occurring in greeting or leave-taking settings did not. Cross-sex touch was more prevalent among adults, whereas same-sex touch was more prevalent when a child was involved. Implications for theoretical perspectives on gender and touch are discussed.