Arg-Gly-Asp (RGD) peptides and the anti-vitronectin receptor antibody 23C6 inhibit dentine resorption and cell spreading by osteoclasts.

Studies with a range of monoclonal and polyclonal antisera to components of the human, rat, and chick vitronectin receptor, alpha V beta 3, and the VLA beta 1 chain show that chick and rat osteoclasts express similar integrin receptors to those described in man. Biochemical analysis with monoclonal antibody 23C6 confirmed the presence on chick osteoclasts of a vitronectin receptor heterodimer of similar size (110/95 kDa reduced) to that immunoprecipitated from human osteoclastoma giant cells. The synthetic peptide GRGDSP, corresponding to the cell adhesion sequence in fibronectin, but not GRGESP peptide, induced significant (P less than 0.005) osteoclast retraction in chick and rat osteoclasts at IC50s (+/- SEM) of 210.0 +/- 14.4 and 191.4 +/- 13.7 microM, respectively; monoclonal anti-vitronectin receptor alpha V beta 3 complex antibody, 23C6, produced similar changes in chick osteoclasts (IC50 = 1.45 +/- 0.22 microM). Antibody 23C6 inhibited the number of pits resorbed in dentine by chick osteoclasts over a concentration range of 4.4 to 88 micrograms/ml; a significant 76% reduction (P = 0.03) was observed at a final concentration of 88 micrograms/ml (6 microM). The effect of peptides upon dentine resorption was less dramatic. No consistent inhibition was seen using chick osteoclasts. Inhibitory effects on resorption by rat osteoclasts were, however, observed; significant reduction in resorption occurred with both GRGDSP (78%; P less than 0.01) and GRGESP (67%; P = 0.02) peptides at 400 microM peptide concentration. These data demonstrate that osteoclast function can be disrupted by low concentrations of the anti-vitronectin receptor antibody, 23C6. The inhibitory effects of the peptides used in this study produced effects on dentine resorption which were generally weaker and variable, although osteoclast cell adhesion was consistently inhibited in an Arg-Gly-Asp (RGD)-dependent manner. We conclude that the vitronectin receptor may play an important role in effecting resorption of mineralized tissues by osteoclasts.     

Retinal responses to white-noise modulated current stimuli

Electric current was injected into a rabbit's eye with white-noise modulations of the current amplitude. A variable D.C. bias was added to the whitenoise stimulus to study the effects of stimulus bias. For each bias level, the ERG response to the electrical stimulus was cross-correlated with the random stimulus to estimate first-and second-order Wiener kernels. The kernels indicated both linear and nonlinear characteristics of the Electrical ERG. The results for the zero biased stimulus are particularly relevant for clinical testing because the root mean square (RMS) level of the stimulus was less than 0.2 mA.This work was supported by NIH Grant Number EY03022     

Activation of Matrix Metalloproteinase-3 is altered at the frog neuromuscular junction following changes in synaptic activity

The extracellular matrix surrounding the neuromuscular junction is a highly specialized and dynamic structure. Matrix Metalloproteinases are enzymes that sculpt the extracellular matrix. Since synaptic activity is critical to the structure and function of this synapse, we investigated whether changes in synaptic activity levels could alter the activity of Matrix Metalloproteinases at the neuromuscular junction. In particular, we focused on Matrix Metalloproteinase 3 (MMP3), since antibodies to MMP3 recognize molecules at the frog neuromuscular junction, and MMP3 cleaves a number of synaptic basal lamina molecules, including agrin. Here we show that the fluorogenic compound (M2300) can be used to perform in vivo proteolytic imaging of the frog neuromuscular junction to directly measure the activity state of MMP3. Application of this compound reveals that active MMP3 is concentrated at the normal frog neuromuscular junction, and is tightly associated with the terminal Schwann cell. Blocking presynaptic activity via denervation, or TTX nerve blockade, results in a decreased level of active MMP3 at the neuromuscular junction. The loss of active MMP3 at the neuromuscular junction in denervated muscles can result from decreased activation of pro-MMP3, or it could result from increased inhibition of MMP3. These results support the hypothesis that changes in synaptic activity can alter the level of active MMP3 at the neuromuscular junction.     

Quinolinone-based agonists of S1P₁: use of a N-scan SAR strategy to optimize in vitro and in vivo activity

We reveal how a N-scan SAR strategy (systematic substitution of each CH group with a N atom) was employed for quinolinone-based S1P(1) agonist 5 to modulate physicochemical properties and optimize in vitro and in vivo activity. The diaza-analog 17 displays improved potency (hS1P(1) RI; 17: EC(50)=0.020 μM, 120% efficacy; 5: EC(50)=0.070 μM, 110% efficacy) and selectivity (hS1P(3) Ca(2+) flux; 17: EC(50) >25 μM; 5: EC(50)=1.5 μM, 92% efficacy), as well as enhanced pharmacokinetics (17: CL=0.15 L/h/kg, V(dss)=5.1L/kg, T(1/2)=24h, %F=110; 5: CL=0.93L/h/kg, V(dss)=11L/kg, T(1/2)=15 h, %F=60) and pharmacodynamics (17: 1.0mg/kg po, 24h PLC POC=-67%; 5: 3mg/kg po, 24h PLC POC=-51%) in rat.     

Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) Serves as a Carbon and Energy Source for a Mixed Culture Under Anaerobic Conditions

We studied the anaerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in a mineral medium by a mixed culture. RDX degradation activity was maintained for more than a year with only the addition of RDX. We observed a steady increase in the protein concentration of the culture from 4.8 μg mL⁻¹ to more than 24.4 μg mL⁻¹, a >400% increase. There was only a slight increase in protein in the RDX unamended control bottles containing live culture, increasing from 4.8 μg mL⁻¹ to 7.8 μg mL⁻¹. Radiolabeled ¹⁴C-RDX confirmed mineralization of the cyclic nitramine to ¹⁴CO₂. After 164 days, 35% of the radiolabel was recovered as ¹⁴CO₂. This is the first report demonstrating the mineralization of RDX when it serves as a growth substrate for a mixed culture.     

Genome-Wide Association Study of Plasma Polyunsaturated Fatty Acids in the InCHIANTI Study

Polyunsaturated fatty acids (PUFA) have a role in many physiological processes, including energy production, modulation of inflammation, and maintenance of cell membrane integrity. High plasma PUFA concentrations have been shown to have beneficial effects on cardiovascular disease and mortality. To identify genetic contributors of plasma PUFA concentrations, we conducted a genome-wide association study of plasma levels of six omega-3 and omega-6 fatty acids in 1,075 participants in the InCHIANTI study on aging. The strongest evidence for association was observed in a region of chromosome 11 that encodes three fatty acid desaturases (FADS1, FADS2, FADS3). The SNP with the most significant association was rs174537 near FADS1 in the analysis of arachidonic acid (AA; p=5.95×10⁻⁴⁶). Minor allele homozygotes had lower AA compared to the major allele homozygotes and rs174537 accounted for 18.6% of the additive variance in AA concentrations. This SNP was also associated with levels of eicosadienoic acid (EDA; p=6.78×10⁻⁹) and eicosapentanoic acid (EPA; p=1.07×10⁻¹⁴). Participants carrying the allele associated with higher AA, EDA, and EPA also had higher low-density lipoprotein (LDL-C) and total cholesterol levels. Outside the FADS gene cluster, the strongest region of association mapped to chromosome 6 in the region encoding an elongase of very long fatty acids 2 (ELOVL2). In this region, association was observed with EPA (rs953413; p=1.1×10⁻⁶). The effects of rs174537 were confirmed in an independent sample of 1,076 subjects participating in the GOLDN study. The ELOVL2 SNP was associated with docosapentanoic and DHA but not with EPA in GOLDN. These findings show that polymorphisms of genes encoding enzymes in the metabolism of PUFA contribute to plasma concentrations of fatty acids.