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PubMed1995

Differential effects of transforming growth factor-beta on osteoclast-like cell formation in mouse marrow culture: relation to the effect of zinc-chelating dipeptides.

The effect of transforming growth factor-beta (TGF-beta) on osteoclast-like cell formation in mouse marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a well-known bone resorbing agent. Osteoclast-like cell formation was estimated with staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of TGF-beta (10(-13)-10(-11) M) caused a significant increase in the number of osteoclast-like multinucleated cells (MNCs); the maximum effect was seen with 10(-12) MTGF-beta. With a higher concentration (10(-10) M) of TGF-beta, the growth factor dramatically inhibited the 1,25-dihydroxyvitamin D5 [1,25(OH)2D3; 10(-8) M]-induced formation of osteoclast-like MNCs. This inhibitory effect was also seen in the formation of osteoclast-like MNCs stimulated by parathyroid hormone (10(-8) M), prostaglandine E2 (10(-6) M), and interleukin-1 alpha (50 U/ml). The stimulatory effect of TGF-beta (10(-12) M) on osteoclast-like MNCs formation was inhibited by zinc sulfate (10(-6) M) or zinc-chelating dipeptide [beta-alanyl-L-histidinato zinc (AHZ), 10(-6) M]. The stimulating effect of TGF-beta was markedly weakened by the presence of EGTA (0.5 mM), a chelator of Ca2+. The inhibitory effect of zinc compounds was not seen in the presence of EGTA. Moreover, the inhibitory effect of TGF-beta (10(-10) M), zinc sulfate (10(-6) M), or AHZ (10(-6) M) on osteoclast-like MNCs formation was not demonstrated in mature osteoclastic cells, although calcitonin (3 x 10(-8) M) significantly inhibited the osteoclastic formation. The present study demonstrates that TGF-beta has a stimulating and an inhibiting effect on osteoclast-like cell formation in mouse marrow culture, and that zinc can inhibit the stimulatory effect of TGF-beta.

Animals↗

PubMed1993

Pharmacology of metabotropic glutamate receptor inhibition of cyclic AMP formation in the adult rat hippocampus.

Phosphoinositide-linked metabotropic glutamate receptors have been well characterized in a variety of CNS tissues. In this study the pharmacology of metabotropic glutamate receptors negatively coupled to cAMP formation was investigated in cross-chopped slices of the adult rat hippocampus. Excitatory amino acid agonists and antagonists were examined for effects on forskolin (30 microM)-simulated cAMP formation. The selective metabotropic glutamate agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and various nonselective metabotropic/ionotropic agonists were found to inhibit forskolin-stimulated cAMP formation. Inhibition of cAMP formation was observed using 1S,3R-ACPD (57% at 100 microM), quisqualate (92% at 500 microM), ibotenate (44% at 500 microM), L-glutamate (41% at 1000 microM), and L-aspartate (59% at 1000 microM). Inhibition of forskolin-stimulated cAMP formation induced by these agonists was observed even in the presence of the ionotropic antagonists MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione. Up to 500 microM of the ionotropic agonists N-methyl-D-aspartate, AMPA, and kainate did not inhibit forskolin-stimulated cAMP formation. L-2-amino-3-phosphonopropionate (500 microM) greatly inhibited the stimulatory effect of 1S,3R-ACPD on phosphoinositide hydrolysis, even in the presence of forskolin. However when measuring cAMP formation, L-2-amino-3-phosphonoproprionate (500 microM) mimicked the effect of 1S,3R-ACPD, producing 64% inhibition of forskolin-stimulated cAMP. These studies show that in the adult rat hippocampus metabotropic glutamate receptors that are negatively linked to cAMP formation have a pharmacology that is distinct from ionotropic glutamate receptors and phosphoinositide-linked metabotropic glutamate receptors.

Alanine↗

PubMed1982

Role of glucose metabolism in acid formation by isolated gastric glands.

The role placed by glucose in providing energy for acid formation was studied in isolated gastric glands from rabbit. The widely-used inhibitors of glycolysis, iodoacetic acid and iodoacetamide were found to inhibit glucose oxidation as well as the indicators of acid formation, respiration and accumulation of aminopyrine. However, the potent inhibitor of acid formation was found to involve a nonspecific mechanism other than the simple inhibition of glycolysis. An alternative approach involved use of the glucose transport inhibitor, phloretin. Phloretin blocked glucose oxidation and also inhibited functional responses. Acid formation was restored easily by the addition of pyruvate or various other oxidizable substrates. Measurement of lactate formation in the absence of exogenous glucose showed that the gastric glands contain very little glycogen. Addition of external glucose resulted in a 10-fold increase in lactate formation and this rate was stimulated further by histamine and rotenone. Rotenone also inhibited both respiration and aminopyrine accumulation; however, the inhibition was not complete. Phloretin treatment resulted in total inhibition of the residual aminopyrine accumulation after rotenone treatment. The results are interpreted to indicate that gastric glands are dependent almost totally on external substrate supply to support acid formation; and, that while anaerobic glucose metabolism can sustain a very low level of acid formation, the major role of glucose is to yield pyruvate equivalents for subsequent oxidation.

Animals↗

PubMed1991

Effect of intracranial pressure on cerebrospinal fluid formation in isolated brain ventricles.

The effect of intracranial pressure on cerebrospinal fluid formation has been studied in cats by ventricular perfusion with the aqueduct of Sylvius blocked (isolated ventricular perfusion). It has been found that intracranial pressure has a considerable effect on the rate of cerebrospinal fluid formation, while increases in pressure cause a significant and prolonged decrease in cerebrospinal fluid formation. The effect was observed in animals whether they were initially perfused under lower or under higher intracranial pressure. Cerebrospinal fluid absorption has been studied under the above conditions and it has been noted that the ventricles are capable of significant cerebrospinal fluid absorption, since in isolated ventricles cerebrospinal fluid formation and absorption were in balance at physiological intracranial pressure. In addition, cerebrospinal fluid formation rate within the isolated brain ventricles has been compared with the formation rate in the whole cerebrospinal fluid system. Since only about 30% of the total cerebrospinal fluid formation was observed by isolated ventricular perfusion, it seems that the brain ventricles are not the exclusive site of cerebrospinal fluid formation.

Animals↗

PubMed1994

Sulphur-containing excitatory amino acid-stimulated inositol phosphate formation in primary cultures of cerebellar granule cells is mediated predominantly by N-methyl-D-aspartate receptors.

The stimulatory effect of excitatory sulphur-containing amino acids on inositol phosphate formation was investigated in primary cultures of cerebellar granule cells. L-Cysteine sulphinate (CSA), L-cysteate (CA), L-homocysteine sulphinate (HCSA), L-homocysteate (HCA) and S-sulpho-L-cysteine (SSC) dose-dependently stimulated the formation of [3H]inositol phosphates exhibiting EC50 values in the range 60-200 microM and maximal effects of six- to 17-fold that of basal [3H]inositol phosphate levels. Endogenous L-glutamate spontaneously released into the extracellular medium or following exposure of cells to HCSA, HCA or SSC did not contribute significantly to formation of [3H]inositol phosphates, whereas 10% of the total [3H]inositol phosphates accumulated following exposure to CSA and CA was due to released L-glutamate. The selective N-methyl-D-aspartate receptor antagonist, D,L-2-amino-5-phosphonopentanoic acid (APV, 500 microM) attenuated by 20% (HCSA) to between 80 and 100% (CSA, CA, SSC, HCA) the formation of [3H]inositol phosphates induced by 1 mM sulphur-containing amino acids. When, however, HCSA was used at 100 microM (a concentration near to its EC50 for phosphoinositide hydrolysis), APV inhibited induced responses by 70%. Sulphur-containing amino acid-stimulated [3H]inositol phosphate formation was unaffected by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM). Inhibition of sulphur-containing amino acid-stimulated [3H]inositol phosphate formation by co-administration of APV and CNQX was similar to that obtained in the presence of APV alone. CSA-, CA-, SSC- and HCA-stimulated [3H]inositol phosphate formation was markedly reduced by removal of Ca2+ from the extracellular medium whereas that stimulated by HCSA was less affected. A similar inhibitory profile was observed when the levels of sulphur-containing amino acid-induced increases in intracellular free calcium ([Ca2+]i) were measured in the presence of 500 microM APV; 1 mM HCSA-induced responses being inhibited by only 30% whereas responses to the remaining sulphur-containing amino acid (also at 1 mM) were inhibited by > 45%. When the sulphur-containing amino acids were used at concentrations approximating their EC50 values for phosphoinositide hydrolysis, APV inhibited the induced increases in [Ca2+]i by 70-100%. HCA and SSC co-administered with the less efficacious but selective metabotropic glutamate receptor agonist, (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) at maximally effective concentrations (1 mM) of each agonist stimulated [3H]inositol phosphate formation in an additive manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

MPP+ and MPDP+ induced oxygen radical formation with mitochondrial enzymes.

MPP+ has been reported to inhibit reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase in mitochondria, which results in the formation of O2(.-). The current report demonstrates that H2O2 and HO. are also products of MPP+ interaction with NADH dehydrogenase. It is possible that MPP. formation precedes the formation of some of these active oxygen species. Reducing equivalents for radical formation come from NADH. MPP+ may be capable of interacting with submitochondrial particles at a site other than the rotenone site, which results in some formation of oxygen radicals. Plasma amine oxidase incubations with MPDP+ resulted in O2.- H2O2, and perhaps HO. formation. This is probably due to MPP. formation from the oxidation of MPDP+. This study presents new findings that indicate the potential importance of oxygen radical formation in mitochondria during MPTP toxicity.

1-Methyl-4-phenylpyridinium↗

PubMed2004

Spectrophotometric determination of de novo hemozoin/beta-hematin formation in an in vitro assay.

Formation of hemozoin in the malaria parasite, due to its unique nature, is an attractive molecular target. Several laboratories have been trying to unravel the molecular mechanism of hemozoin biosynthesis within the parasite digestive vacuoles. Use of different assay protocols for in vitro beta-hematin (synthetic identical to hemozoin) formation by these laboratories has led to inconsistent and often contradictory findings. Much of the difficulty may be attributed to oligomeric heme aggregates, which may be indistinguishable in some detection approaches if adequate separation of beta-hemtin is not achieved. Therefore, there is an urgent need for a widely accepted protocol for in vitro beta-hematin formation. We describe here a spectrophotometric assay for in vitro beta-hematin formation. The assay has been validated with the Plasmodium falciparum lysate, the parasite lipid extracts, and some commercially available fatty acids, which are known to initiate/catalyze beta-hematin formation in vitro. The necessity for multiple wash steps for accurate quantification of de novo hemozoin/beta-hematin formation was verified experimentally. It was necessary to wash the pellet, which contains beta-hematin and heme aggregates, sequentially with Tris/SDS buffer and alkaline bicarbonate solution for complete removal of monomeric heme and heme aggregates and accurate quantification of beta-hematin formed during the assay. The pellets and side products in the supernatant were characterized by infrared spectroscopy. No beta-hematin formation occurred in the absence of a catalytic/initiating factor. Based on these findings, a filtration-based assay that uses 96-well microplates, and which has important application in in vitro screening and identification of novel inhibitors of hemozoin formation as potential blood schizontocidal antimalarials, has been developed.

Animals↗

PubMed2005

Provider response to different formats of the adult immunization schedule.

BACKGROUND: Providers' failure to administer vaccines in accordance with established recommendations is a well-recognized barrier to national immunization efforts. This study evaluated the ease of use of two different formats of the Centers for Disease Control and Prevention's (CDC) adult immunization schedule by physicians in private practice, where the majority of adult immunizations are administered. METHODS: A series of focus groups was conducted with 94 physicians and other clinical staff in 11 private practices (family medicine and internal medicine) in six U.S. cities. Each session was based on a structured set of questions that explored barriers to adult immunizations, followed by three mock clinical scenarios to examine how each of two graphical depictions of the 2003-2004 adult immunization schedule (one from the CDC's Advisory Committee on Immunization Practices, and the other from the Immunization Action Coalition) might facilitate assessments of recommended immunizations. Group dialogue and individual participants' written responses to the scenarios and the alternate schedule formats were analyzed. RESULTS: Providers perceived multiple barriers to adult immunization independent of immunization schedule formats, chiefly patients' low interest in immunization and refusal of vaccines. Most participants were not familiar with either format of CDC's adult immunization schedule before the study, but quickly developed strong preferences for one versus the other (usually the second format that they encountered). About half of the providers changed their vaccine recommendations for clinical scenarios when they consulted either schedule format, although some of the changes were not clinically appropriate. Participants suggested several ways to enhance the availability of the information contained in the schedule formats, especially through electronic means. CONCLUSIONS: This qualitative study suggests ways in which graphic depictions of an adult immunization schedule may address adult immunization barriers. Greater provider familiarity with schedule formats will be critical to their appropriate application in clinical encounters.

Adult↗

PubMed2006

Fluorescence-coupled CD conformational monitoring of filament formation of tau microtubule-binding repeat domain.

To clarify the contribution of the three- or four-repeated peptide moiety in tau microtubule-binding domain (MBD) to paired helical filament (PHF) formation, conformational transition accompanied by heparin-induced filament formation was investigated stepwise for four repeat peptides (R1-R4), one three-repeated R1-R3-R4 peptide (3RMBD), and one four-repeated R1-R2-R3-R4 peptide (4RMBD) using a combination of thioflavin S fluorescence and circular dichroism (CD) measurements in a neutral buffer (pH 7.6). The comparison of the fluorescence profile of each repeat peptide with those of 3RMBD and 4RMBD showed the synergistic contribution of R1-R4 to PHF formation of MBD. The CD spectrum measured as a function of filament formation time indicates that: (i) two conformational transitions occur for the filament formations of R3 (from the random structure to the beta-sheet structure) and 3RMBD (from the random structure to the alpha-helix structure), (ii) the filament formations of R2 and 4RMBD proceed via the synchronized conformational transitions of the alpha-helix and random structures, and (iii) the filament formation of 4RMBD is dependent on the aggregation behavior of R2. These data are useful for elucidating the MBD conformational transition in tau PHF formation.

Amino Acid Sequence↗

PubMed2004

Inhibitory effect of beta-cryptoxanthin on osteoclast-like cell formation in mouse marrow cultures.

The carotenoid beta-cryptoxanthin has been shown to have an inhibitory effect on bone-resorping factor-stimulated bone resorption in rat bone tissues in vitro. The effect of beta-cryptoxanthin on osteoclast-like cell formation in mouse marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a bone-resorbing agent [parathyroid hormone (1-34) (PTH), prostaglandin E(2), 1,25-dihydroxyvitamin D(3), lipopolysaccharide, or tumor necrosis factor-alpha (TNFalpha)] with an effective concentration. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. The presence of PTH (10(-7)M), prostaglandin E(2) (10(-5)M), 1,25-dihydroxyvitamin D(3) (10(-7)M), lipopolysaccharide (10 microg/mL), or TNFalpha (10 ng/mL) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited in the presence of beta-cryptoxanthin (10(-8) to 10(-6)M). beta-Cryptoxanthin (10(-7) and 10(-6)M) significantly inhibited dibutyryl cyclic adenosine monophosphate (DcAMP) (10(-5)M) or phorbol 12-myristate 13-acetate (PMA) (10(-5)M), an activator of protein kinase C, induced osteoclast-like cell formation. Also, beta-cryptoxanthin (10(-7) and 10(-6)M) had a significant inhibitory effect on osteoclast-like formation induced by receptor activator of NF-kappaB ligand (RANKL) (10 and 20 ng/mL) in the presence of macrophage colony-stimulating factor (M-CSF) (10 and 20 ng/mL). The stimulatory effect of RANKL and M-CSF on osteoclast-like cell formation was significantly enhanced in the presence of PMA, while such an effect was not seen by DcAMP. beta-Cryptoxanthin (10(-6)M) significantly inhibited osteoclast-like cell formation induced by RANKL and M-CSF in the presence of PMA or DcAMP. Moreover, the inhibitory effect of beta-cryptoxanthin on RANKL plus M-CSF-, PTH-, or TNFalpha-induced osteoclast-like cell formation was not observed in the presence of cycloheximide (10(-7)M), an inhibitor of protein synthesis at translational process, or 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (10(-6)M), an inhibitor of transcription. This study demonstrates that beta-cryptoxanthin has a potent inhibitory effect on osteoclast-like cell formation in mouse marrow culture. The inhibitory action of beta-cryptoxanthin may partly involve in a newly synthesized protein component which is related to RANKL stimulation in osteoclastogenesis.

Animals↗

PubMed2005

Ectopic bone formation in rats: the importance of the carrier.

Much research has been done to develop the ideal bone graft substitute (BGS). One approach to develop this ideal BGS is the use of growth factors, but for this approach osteoprogenitor cells are needed at the site of reconstruction. An alternative is a cell-based approach, where enough cells are provided to form bone in a carrier material. In previous studies of our group, titanium (Ti) carriers have been used, because of the excellent mechanical properties and the bone-compatibility of this material. On the other hand, calcium phosphate (CaP) ceramics are known for their excellent osteoconductivity. The aim of this study is to investigate the influence of the carrier in a cell-based bone regeneration approach, whereby we hypothesize that CaP-ceramic implants will induce more bone formation than Ti-fiber implants, in the same animal model as our previous experiment. Ti-fiber mesh implants and ceramic implants were seeded with rat bone marrow cells (RBM) and implanted subcutaneously. Histological analysis after one, three and six weeks showed differences in the way of bone formation in the two groups: bone appeared to grow from the center to the periphery of the implant in the titanium group, while bone formation in the ceramic group occurred through the whole implant. Histomorphometrical analysis after one week showed very limited bone formation for both the titanium and ceramic group. At three weeks, the amount of bone formation was increased till about 10% for the titanium group and 18% for the ceramic group. No significant difference between the two groups could be observed. In the six week group, the bone formation was 6% (Ti) and 23% (CaP), respectively (P < 0.001). Further, bone formation started earlier in the CaP-ceramic scaffolds than in the Ti scaffolds. Our hypothesis could be confirmed: ceramic implants induce more bone formation than titanium implants.

Animals↗

PubMed2005

Nell-1 induced bone formation within the distracted intermaxillary suture.

Maxillary bone deficiencies, such as cleft palate and underdeveloped maxilla that require bone graft or regeneration after orthopedic or surgical expansion, pose a significant biomedical burden. Nell-1 is a secreted molecule that possesses chordin-like domains and induces cranial suture bone growth and osteoblast differentiation. To accelerate bone formation in acutely distracted palatal sutures, rat organ cultures were stimulated with Nell-1 or BMP-7 for 8 days in vitro. We hypothesized that Nell-1 stimulation to the distracted palatal suture would accelerate bone formation. Distracted palates of 4-week-old male rats were maintained in an organ culture system, and tissue was either unstimulated or stimulated with Nell-1 or BMP-7 for 8 days. MicroCT was conducted to quantitate bone formation, while alcian blue staining was conducted for cartilage localization. Immunohistochemistry of Sox9 for chondrocyte proliferation, type X collagen for hypertrophic cartilage in endochondral bone formation, and bone sialoprotein for bone formation was conducted to characterize the cellular mechanism of newly developed tissues. Distracted palates cultured in the presence of Nell-1 or BMP-7 produced statistically significantly (P < 0.05) more bone and cartilage within the intermaxillary suture, relative to unstimulated control samples. While both BMP-7 and Nell-1 induced similar bone formation in the distracted suture, BMP-7 induced both chondrocyte proliferation and differentiation, while Nell-1 accelerated chondrocyte hypertrophy and endochondral bone formation. While both Nell-1 and BMP-7 are effective in forming bone in the distracted palatal suture, they are suggested to have distinctively different mechanisms. The ability of Nell-1 to accelerate bone formation within the palate suture demonstrates the versatility of Nell-1 within the craniofacial complex as well as an exciting advance in palate suture defect healing.

Animals↗

PubMed2004

Intragastric formation and modulation of N-nitrosodimethylamine in a dynamic in vitro gastrointestinal model under human physiological conditions.

Human exposure to carcinogenic N-alkylnitrosamines can occur exogenously via food consumption or endogenously by formation of these compounds through nitrosation of amine precursors. Information on the intragastric formation of NDMA from complex mixtures of precursors and inhibitors in humans is not available. In this study the formation of N-nitrosodimethylamine (NDMA) has been quantitatively analysed in a dynamic in vitro gastrointestinal model, in which gastric conditions can be modulated and closely simulates the physiological situation in humans. Substantial amounts of NDMA were produced when nitrite and dimethylamine or codfish were simultaneously introduced into the model. However, humans are gradually exposed to nitrite by the intake of nitrate-containing food. Nitrate secreted in saliva is converted to nitrite by oral bacteria. To mimic the human exposure to nitrite in a realistic way, nitrite was gradually added into the gastric compartment, simulating the swallowing of nitrite containing oral fluid after the intake of nitrate at the level of 0.1-10 times the ADI. Under these conditions, the cumulative amounts of NDMA formed were 2.3-422 microg NDMA and 1.8-42.7 microg NDMA at a rapid and slow gastric pH decrease, respectively. Beside codfish, various fish species and batches in combination with nitrite, simulating the intake of for times the ADI of nitrate, were investigated. Herring, pollack and plaice were also able to induce NDMA formation. Mackerel, salmon and pike perch did not result in increased NDMA formation. Furthermore, the effect of nitrosation modulators on NDMA formation was investigated. Thiocyanate (2 mM) increased NDMA formation, but the increase was not statistically significant. In contrast, orange jus and tea effectively, but not totally, reduced the amount of NDMA formed in the gastric compartment. These experiments show that (1). the dynamic in vitro gastrointestinal model is an appropriate tool for mechanistic studies on the intragastric formation of nitrosamines, and (2). that the results obtained with this model are helpful in evaluating human cancer risk for the combined intake of codfish-like fish species and nitrate-containing vegetables.

Animals↗

PubMed2006

Staphylococcus aureus capsular material promotes osteoclast formation.

Osteomyelitis, which is most frequently due to infection by Staphylococcus aureus, commonly causes bone destruction. S. aureus is known to secrete a number of surface-associated proteins that are potent stimulators of bone resorption. The precise cellular and humoral mechanisms that mediate this stimulatory effect are uncertain. In this study, we have determined whether osteoclast formation and resorption is directly promoted by surface-associated proteins. Surface-associated material (SAM) obtained from a 24-hour culture of S. aureus was added to cultures of mouse and human monocytes. Human monocyte cultures were incubated in the presence and absence of a soluble receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). In cultures where M-CSF, RANKL, and SAM were added together, osteoclast formation did not exceed that seen in cultures with M-CSF and RANKL. In keeping with this finding, SAM did not increase osteoclast formation and resorption when mouse monocytes were cocultured with RANKL-expressing osteoblasts. In the absence of RANKL, however, SAM was capable of inducing osteoclast formation in cultures of human monocytes. This finding was evidenced by the generation of vitronectin receptor and tartrate-resistant acid phosphatasepositive multinucleated cells that were capable of lacunar resorption. Inhibitors of RANKL-dependent (RANK:Fc, OPG) and RANKL-independent (anti-TNF-alpha, gp130, IL-8, TGF-beta) osteoclast formation did not inhibit SAM-induced osteoclast formation. SAM did not stimulate mature osteoclast resorption activity. These findings indicate that RANKL, which is present in the circulation as a soluble factor, does not play a role in osteoclast formation in the presence of S. aureus SAM and that S. aureus SAM contains a soluble factor that promotes osteoclast formation by a RANKL-independent mechanism.

Animals↗

PubMed2004

Experimental and finite element analysis of the rat ulnar loading model-correlations between strain and bone formation following fatigue loading.

The rat forelimb compression model has been used widely to study bone response to mechanical loading. We used strain gages to assess load sharing between the ulna and radius in the forelimb of adult Fisher rats. We used histology and peripheral quantitative computed tomography (pQCT) to quantify ulnar bone formation 12 days after in vivo fatigue loading. Lastly, we developed a finite element model of the ulna to predict the pattern of surface strains during compression. Our findings indicate that at the mid-shaft the ulna carries 65% of the applied compressive force on the forelimb. We observed large variations in fatigue-induced bone formation over the circumference and length of the ulna. Bone formation was greatest 1-2 mm distal to the mid-shaft. At the mid-shaft, we observed woven bone formation that was greatest medially. Finite element analysis indicated a strain pattern consistent with a compression-bending loading mode, with the greatest strains occurring in compression on the medial surface and lesser tensile strains occurring laterally. A peak strain of -5190 microepsilon (for 13.3N forelimb compression) occurred 1-2 mm distal to the mid-shaft. The pattern of bone formation in the longitudinal direction was highly correlated to the predicted peak compressive axial strains at seven cross-sections (r2 = 0.89, p = 0.014). The in-plane pattern of bone formation was poorly correlated to the predicted magnitude of axial strain at 51 periosteal locations (r2 = 0.21, p < 0.001), because the least bone formation was observed where tensile strains were highest. These findings indicate that the magnitude of bone formation after fatigue loading is greatest in regions of high compressive strain.

Animals↗

PubMed2005

Prediction of heat of formation and related parameters of high energy materials.

Heat of formation is one of the most important parameters in the performance prediction of explosive and propellant formulations and their individual ingredients. This paper reports the development of user-friendly computer code for the prediction of heat of formation based on two approaches. In first methodology, the logic of Benson's Group additivity method and in the second method, the logic of Pedley method was used for predicting the heats of formation of high energy materials (HEMs). The predicted heats of formation by Benson method for various classes of high energy materials gave deviation in the range of 2-10%, whereas nearly 10-15% deviation was observed using Pedley methodology in comparison to experimental values. The linear regression coefficient values (R(2)) of 0.9947 and 0.9637 are obtained for heat of formation values predicted by this code using methodologies I and II, respectively. The newly developed code LOTUSES (version 1.3) has been validated by calculating the heats of formation of standard explosives such as TNT, pentaerythritol tetranitrate (PETN), RDX, HMX, etc., To the best of our knowledge, no such code is reported in literature which can predict heats of formation values integrated with performance parameters of HEMs belonging to all categories of organic compounds viz. aliphatic, aromatic and heterocyclic materials. The code can also be used to obtain parameters such as velocity of detonation, C-J pressure, volume of explosion products, power index, temperature of explosion and oxygen balance of HEMs. The code has been developed in Visual Basic having enhanced Windows environment. This software namely LOTUSES 1.3 is an updated version of the earlier ones namely LOTUSES 1.1 and 1.2 which do not cater for the calculation of heat of formation and temperature of explosion of HEMs. LOTUSES 1.3 is, therefore, a totally integrated software for computing most of the vital parameters of HEMs requiring mainly the molecular structural information of an explosive under consideration.

Energy-Generating Resources↗

PubMed2004

Aging exacerbates neointimal formation, and increases proliferation and reduces susceptibility to apoptosis of vascular smooth muscle cells in mice.

OBJECTIVES: In response to injury, aging mediates exaggerated neointimal formation, the pathologic hallmark of obliterative vascular diseases. We assessed the development of neointima in a model of mechanical vascular injury in aging mice (18 months old) and young mice (2 months old). To investigate the mechanisms by which aging affects neointimal formation, we also carried out a set of in vitro studies to characterize the biologic properties of vascular smooth muscle cells (VSMCs) derived from aging and young mice. METHODS: Aging and young mice were subjected to wire injury to the carotid artery. Four weeks later injured arteries were harvested, and neointimal formation was histologically assessed. The profiles of angiogenesis-related genes between aortic VSMCs derived from aging and young mice were compared with complementary DNA arrays. Expression of platelet-derived growth factor receptor-alpha (PDGFR-alpha) and proliferation in response to platelet-derived growth factor-BB (PDGF-BB) by VSMCs were assessed. Susceptibility to apoptosis in aging and young VSMCs in response to nitric oxide and serum starvation was investigated. In addition, the level of apoptosis in neointimal VSMCs (by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay) was compared between aging and young animals. RESULTS: When compared with young mice, aging mice exhibited exaggerated neointimal formation (intima-media ratio, 1.17 +/- 0.57 vs 0.49 +/- 0.16; P < .0001). Aging VSMCs expressed higher levels of PDGFR-alpha (12.0% +/- 2.7% vs 3.2 +/- 0.67%; P = .034) and greater proliferative response (4-fold increase) to PDGF-BB, compared with young VSMCs. However, aging VSMCs were less susceptible to apoptosis when subjected to serum starvation (75% less) and exposure to nitric oxide (50% less). Furthermore, there was more apoptosis in the neointima of young arteries than in their aging counterparts (8.75% +/- 3.3% vs 2.8% +/- 1.9; P = .021). CONCLUSIONS: Age-dependent increases in PDGFR-alpha may alter VSMC proliferation, and when coupled with resistance to apoptosis could contribute to exaggerated neointima formation in aging animals. Of significance, our findings in the mouse will enable application of abundant molecular tools afforded by this species to further dissect the mechanisms of exaggerated neointimal formation associated with aging. CLINICAL RELEVANCE: Neointimal formation is the pathologic hallmark of obliterative vascular diseases, including primary atherosclerosis, post stent restenosis, graft occlusion after vascular bypass procedures, and transplant allograft vasculopathy. Aging is an independent risk factor for development of cardiovascular diseases, and aging exaggerates neointimal formation after vascular injury. Understanding the mechanisms responsible for this phenomenon may facilitate prevention or provide new therapies for vascular occlusive diseases, which are so prevalent in the aging population. Our ability to reproduce the model in the mouse will no doubt facilitate such understanding.

Aging↗

PubMed2005

Modulatory effect of glutathione status and antioxidants on methylmercury-induced free radical formation in primary cultures of cerebral astrocytes.

Excessive free radical formation has been implicated as one of the causative factors in neurotoxic damage associated with variety of metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-dependent neurotoxicity remains far from clear, overwhelming data give credence to a mediatory role for astrocytes, a major cell type that preferentially accumulates MeHg. To extend our recent findings of MeHg-induced increase in ROS formation (G. Shanker, J.L. Aschner, T. Syversen et al., Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury, Mol. Brain Res. 128 (2004) 48-57), the present studies were designed to assess the effect of modulating intracellular glutathione (GSH) content, on ROS generation, in the absence and presence of MeHg. Intracellular GSH was reduced by treatment with 100 microM buthionine-L-sulfoxane (BSO) for 24 h, and increased by treatment with 1 mM l-2-oxothiazolidine-4-carboxylic acid (OTC) for 24 h. Additionally, the effects of the selective antioxidants, catalase (1000 U/ml for 1 h), an H2O2 scavenger, and n-propyl gallate (100 microM for 1 h), a superoxide radical (*O2-) and possibly hydroxyl radical (*OH) scavenger on MeHg-induced ROS formation were examined. After these treatments, astrocytes were exposed to +/-10 microM MeHg for 30 min, following which the fluorescent probes, CM-H2DCFA and CM-H2XRos were added; 20 min later, laser scanning confocal microscopy (LSCM) images were obtained. Exposure of astrocytes for 24 h to 100 microM BSO, a GSH synthesis inhibitor, led to a significant increase in mitochondrial ROS (i.e., *O2-, *NO, and ONOO-) formation, as assessed with CM-H2XRos mitotracker red dye. Similarly, BSO increased ROS formation in various intracellular organelles, as assessed with CM-H2DCFDA. BSO in combination with MeHg increased fluorescence levels in astrocytes to levels above those noted with BSO or MeHg alone, but this effect was statistically indistinguishable from either of these groups (BSO or MeHg). Pretreatment of astrocytes for 24 h with 1 mM OTC abolished the MeHg-induced increase in ROS. Results similar to those obtained with OTC were observed with the free radical scavenger, n-propyl gallate (n-PG). The latter had no significant effects on astrocytic fluorescence when administered alone. This *O2- and possibly *OH radical scavenger significantly attenuated MeHg-induced ROS formation. Catalase, an H2O2 scavenger, was less effective in reducing MeHg-induced ROS formation. Taken together, these studies point to the important protective effect of adequate intracellular GSH content as well as antioxidants against MeHg-triggered oxidative stress in primary astrocyte cultures.

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