Adenosine amine congener as a cochlear rescue agent.

UNLABELLED: We have previously shown that adenosine amine congener (ADAC), a selective A1 adenosine receptor agonist, can ameliorate noise- and cisplatin-induced cochlear injury. Here we demonstrate the dose-dependent rescue effects of ADAC on noise-induced cochlear injury in a rat model and establish the time window for treatment. METHODS: ADAC (25-300 µg/kg) was administered intraperitoneally to Wistar rats (8-10 weeks old) at intervals (6-72 hours) after exposure to traumatic noise (8-16 kHz, 110 dB sound pressure level, 2 hours). Hearing sensitivity was assessed using auditory brainstem responses (ABR) before and 12 days after noise exposure. Pharmacokinetic studies investigated ADAC concentrations in plasma after systemic (intravenous) administration. RESULTS: ADAC was most effective in the first 24 hours after noise exposure at doses >50 µg/kg, providing up to 21 dB protection (averaged across 8-28 kHz). Pharmacokinetic studies demonstrated a short (5 min) half-life of ADAC in plasma after intravenous administration without detection of degradation products. CONCLUSION: Our data show that ADAC mitigates noise-induced hearing loss in a dose- and time-dependent manner, but further studies are required to establish its translation as a clinical otological treatment.

Mechanisms of focal calcification in rabbit aortas: role of calcifying vesicles.

BACKGROUND: We previously demonstrated that calcification in rabbit thoracic aortas is initiated in the lower zone of the extensively thickened plaques (LZP) adjacent to the media. Whether osteogenesis or a local increase in calcifying vesicles underlies the focal calcification remains to be established. METHODS: To determine that focal calcification is related to osteogenesis, an obligatory osteogenic biomarker, alkaline phosphatase (ALP) activity, was evaluated in the unfixed thoracic aorta sections of rabbits fed a supplemental cholesterol diet and of humans with advanced atherosclerosis. To determine whether blood shortage to the smooth muscle cells (SMCs) imposed by intima thickening may increase calcifying vesicles, the effect of serum deprivation on the biogenesis of calcifying vesicles in cultured SMCs was investigated. RESULTS: (1) In contrast to positive rabbit kidney cross sections and consistent with the activity in various isolated subcellular fractions of aortas, ALP activity was absent in the media, adventitia, lesions, and LZP of rabbit aortas or in the fibrointima of human aortas. (2) Histologic assessments of the lesions indicate the absence of bone cells or osteoid. (3) Depletion of the serum from culture media caused a twofold increase in the levels of ALP-deficient and adenosine triphosphatase-rich calcifying vesicles, which were released from the cells by treatment with 0.05% trypsin-0.02% ethylenediaminetetraacetic acid for 15 minutes. CONCLUSIONS: (1) Focal calcification in rabbit aortas and diffuse mineralization in human fibrointima are not associated with osteogenesis, and (2) blockade of the blood supply to SMCs may trigger the cells to produce more calcifying vesicles, thereby leading to site-specific calcification in rabbit aortas.

Accumulation of low density lipoprotein associated cholesterol in calcifying vesicle fractions correlates with intimal thickening in thoracic aortas of juvenile rabbits fed a supplemental cholesterol diet.

BACKGROUND: It has been shown that calcifying vesicles play an important role in aortic calcification and that cholesterol content in the isolated vesicle fraction is increased when rabbits are fed supplemental cholesterol diets. Whether lipoprotein-associated cholesterols and other lipids are also increased in the vesicle fraction and whether the increase correlates with atherosclerosis remain unknown. RESULTS: Fourteen juvenile male rabbits fed an atherogenic diet containing 0.5% cholesterol and 2% peanut oil for 3 months developed varying degrees of hypercholesterolemia and intimal thickening in the ascending thoracic aorta. The correlation between these two parameters was insignificant, and likely attributable to the use of small numbers of rabbits in this study. Despite this lack of correlation, we demonstrate that the accumulation of cholesterol in calcifying vesicle fractions obtained from the collagenase-digested aorta fragments correlates well with intimal thickening (r2 = 0.98, p < 0.0001). To a smaller degree, the correlation was also significant between intimal thickening and the cholesterol accumulation in the microsomal and post-vesicle fractions. The cholesterol supplemental diet increased the low density lipoprotein-cholesterol (LDL-C) content in calcifying vesicle fractions by 3-fold but did not affect the triglyceride content. High density lipoprotein-cholesterol (HDL-C) and very low-density lipoprotein cholesterol (VLD-C) were absent in calcifying vesicle fractions. CONCLUSION: When limited numbers of rabbits are used, LDL-C accumulation in calcifying vesicle fractions is a better biomarker for atherosclerosis than LDL-C levels in the serum. The close association of LDL-C with calcifying vesicles may play an important role in atherosclerosis and calcification.

Effects of dietary calcium on atherosclerosis, aortic calcification, and icterus in rabbits fed a supplemental cholesterol diet.

BACKGROUND: Vascular calcification is implicated in myocardial infarction, instability and rigidity of the aortic wall, and bioprosthetic failures. Although an increase in the calcium (Ca) content in atherogenic diets has been shown to decrease atherosclerosis in rabbits, whether Ca supplementation and deficiency can affect atherosclerosis-related aortic calcification remains unknown. RESULTS: New Zealand White male rabbit littermates were fed an atherogenic diet containing 0.5% cholesterol and 2% peanut oil. The Ca content of the diet, which normally contains 1%, was adjusted to 0.5 or 3%. Segments of thoracic aortas were dissected from rabbits for histological evaluations and Ca and Pi determinations. Rabbits with calcium supplementation were maintained for 4 months, whereas those with calcium deficiency were maintained for 2 1/2 months due to severe icterus beyond this stage. The ratios of intimal to medial areas and calcified to intimal areas were used to semi-quantify lesion accumulation and calcification, respectively. Icterus was estimated from the extent of yellowing of the skin, sclera, and mucous membranes along with gross evidence of hepatic lipidosis and/or biliary obstructions. Statistical analysis of 16 matched littermates shows that Ca supplementation significantly decreased the lesions by 41% (p < 0.05) and markedly inhibited calcification by 62% (p < 0.05). Statistical analysis of 11 matched littermates shows that Ca deficiency significantly increased the lesions by 2.7-fold (p < 0.05) and that the diet caused a small but significant calcification not seen in the sibling groups with normal dietary Ca. Ca supplementation caused a significant 30% decrease in serum cholesterol (p < 0.05). Calcium deficiency increased serum cholesterol by 57% (p < 0.001). Serum cholesterol and LDL-cholesterol levels in Ca deficient rabbits were 2-fold higher than those with high Ca diets. Ca supplementation decreased soluble Ca and Pi content in aortas, suggesting that this effect may underlie the effects of Ca supplementation on calcification. Calcium deficiency increased icterus by 33% (p < 0.05), which may affect hepatic clearance of cholesterol, while calcium supplementation decreased it by 43% (p < 0.001). CONCLUSION: Ca supplementation to an atherogenic diet inhibits atherosclerosis, aortic calcification, and icterus, whereas a Ca deficient-diet promotes them.

Role of bicarbonate/CO2 buffer in the initiation of vesicle-mediated calcification: mechanisms of aortic calcification related to atherosclerosis.

Calcifying vesicles play an important role in the mechanism of aortic calcification induced by dietary cholesterol interventions. This study was initiated to test the hypothesis that alterations in the ratio of bicarbonate/CO2, which is a main physiological buffer, could affect vesicle-mediated calcification. Using rabbits as a model, in vitro calcification of vesicles isolated from aortas was performed to study the effect of the bicarbonate buffer on the mineralization process. When Tris buffer was initially used to maintain pH of the media, ATP-dependent vesicle calcification increased with pH of calcifying media. By replacing Tris with physiological bicarbonate/CO2 buffer, ATP-dependent vesicle calcification increased rapidly with increased ratios of bicarbonate/CO2. The increase appears to be a result of elevated levels of pH through the alteration in the ratios of bicarbonate/CO2. The effect of the physiological concentration of bicarbonate (30 mM) on pH of calcifying media was remarkable since 50 mM of Tris buffer at pH 7.6 failed to prevent a rapid rise in pH under atmospheric CO2. The effect of bicarbonate and CO2 on vesicle calcification was dependent on the ratio of the surface area to the volume of calcifying media, since the ratio profoundly affects the exchange rate between the gas and liquid phases of CO2. Although the pathological conditions that alter the pH remain unknown, it is conceivable that blockage in the supply of blood CO2 to the media by intimal thickening in the lesions could contribute to focal calcification. We conclude that bicarbonate buffer could provide a dynamic and rapid transitional increase in pH of extracellular fluids, thereby creating a favorable condition for the initiation of vesicle-mediated calcification under pathological conditions.

Mechanism of dystrophic calcification in rabbit aortas: temporal and spatial distributions of calcifying vesicles and calcification-related structural proteins.

INTRODUCTION: We have previously demonstrated the accumulation of calcifying vesicles in the thoracic aorta undergoing dystrophic calcification. Whether the distributions of other structural proteins related to calcification such as collagen and elastin fibers undergo coordinated modifications has not been established. METHODS: Young rabbits with various degrees of aortic calcification induced by cholesterol dietary interventions were used as an animal model to study the correlations. RESULTS: Rabbits fed a diet enriched in cholesterol for 3 months accumulated calcifying vesicles in the ascending thoracic aortas but did not develop histologically identifiable calcification. There were concomitant marked thickenings of the intima with focal deposition of collagen and disruption of the internal elastic fibers at this stage. By the 6th month, calcification was predominantly present in the intimal area adjacent to the media. At this calcified stage, calcifying activity of vesicles was higher than earlier stages. Concomitantly, collagen deposition in the lesions was intensified and the internal elastic fibers were completely disintegrated. These changes were found to be more profound in the proximal than in the distal portion of the aortas. CONCLUSION: The coordinated changes in the accumulation of collagen, disintegration of internal elastic fibers, and the appearance of calcifying vesicles in the lesions before calcification may set the stage for aortic calcification.

In vitro effect of cholesterol on calcifying activity of vesicles isolated from rabbit aortas.

It has been shown that vesicles play a key role in the onset mechanism of aortic calcification related to cholesterol-induced atherosclerosis. This study using a rabbit model was conducted to determine whether cholesterol exerts a direct effect on vesicle's calcifiability. Inclusion of cholesterol in calcifying media stimulated ATP-initiated deposition of calcium in a dose-dependent manner by vesicles isolated from normal aortas using crude collagenase digestion. By contrast, cholesterol did not significantly affect ATP-promoted calcification if vesicles were isolated from atherosclerotic aortas. To determine whether high cholesterol levels in atherosclerotic vesicle preparations may have already maximized calcifying activity and therefore account for lack of the vesicle's response to the sterol, Fourier transform infrared spectroscopy (FT-IR) was used to compare the cholesterol contents in control and atherosclerotic vesicles. The spectral patterns revealed higher levels of cholesterol in vesicle preparations from atherosclerotic aortas than those from normal aortas. Removal of extra-vesicular cholesterol micelles from atherosclerotic vesicles by a relatively low centrifugal force sensitized the vesicles to cholesterol stimulation causing a 2-fold increase in calcifying activity. Of various oxidized forms of cholesterol tested, 7-keto and 6-keto cholesterol enhanced the activity by 2-fold. Altogether, these observations suggest that cholesterol and especially its oxidized forms may induce aortic calcification by directly enhancing the vesicle's ability to calcify.

Mechanisms of calcification by vesicles isolated from atherosclerotic rabbit aortas.

Although several lines of evidence support the role of calcifiable vesicles in dystrophic vascular calcification, the mechanisms whereby vesicles promote aortic calcification remain incompletely understood. Previous reports indicate that ATP promotes in vitro vesicle calcification. Whether ATP-initiated calcification is simply mediated through increased Pi concentrations or by other unknown mechanisms related to ATP hydrolysis is unclear. To determine whether high Pi levels resulting from ATP hydrolysis may cause CaxP ion products to surpass the threshold for calcium phosphate precipitation, 3 mM Pi instead of 1 mM ATP was added to calcifying media. The inclusion of 1 mM ATP in calcifying media with an initial serum level of Ca2+ (1.45 mM) and Pi (2.3 mM) was much more effective in promoting calcification than the addition of 3 mM Pi. The higher effectiveness of ATP over Pi in promoting calcification was consistent throughout various incubation periods and vesicle protein ranges. To minimize the effect of CaxPi ion products on calcification, the ion product was kept within the physiological ranges throughout the incubation period by reducing initial Pi or ATP concentrations in calcifying media. At these low levels of ion products, ATP was still more effective than Pi in promoting calcification. Both ATP- and Pi-stimulated calcifications were found to increase with increasing levels of ion products whereas greater effectiveness of ATP over Pi remained unaltered. These observations indicate that ATP hydrolysis may initiate calcification through some mechanisms other than a simple provision of Pi in order to surpass the solubility products. Concanavalin A (Con A) was found to bind to vesicles and to enhance both ATP- and Pi-promoted calcification. Taken together, these observations suggest that ATP hydrolysis, CaxP ion products, and vesicle-associated carbohydrates are implicated in vesicle-mediated calcification.

Induction of calcification in rabbit aortas by high cholesterol diets: roles of calcifiable vesicles in dystrophic calcification.

Atherosclerotic calcification may weaken the aorta wall and thereby lead to rupture of the vessel. The mechanism whereby aortas undergo calcification remains unclear. Previous reports in this laboratory showed that, after 2 months of cholesterol-supplemental feeding, an increase in calcifiability of membrane vesicles isolated from rabbit aortas precedes substantial arterial calcification. Further, the mineral was deposited by isolated calcifiable vesicles as an amorphous phase similar to minerals in human aortas at an early stage of atherosclerosis. In the current study, atherosclerotic calcification was induced by exposing rabbits to a 1% cholesterol-rich diet for 3 or 6 months. After 3 months of dietary interventions, atherosclerotic lesions were fully developed. Fatty streaks were evident in areas proximal to the heart and became less frequent in the distal areas. However, calcification was not yet identifiable histologically or by using Fourier transform spectroscopy (FT-IR). After 6 months of high cholesterol treatment, aortas were partially calcified. Histochemical staining for mineral revealed that calcification appeared to occur predominantly in the intimal areas immediately adjacent to the media. Fourier Transform Imaging analysis demonstrated that the mineral deposited in atherosclerotic rabbit aortas was a hydroxyapatite-like phase. To determine whether aorta vesicles play a role in mineral formation in aortas, vesicles were isolated from calcified aortas and then their calcifiability was compared to that in normal vesicles. Interestingly, during the course of vesicle isolation, we found that calcifiable vesicles with much higher calcifiability than normal vesicles could be readily isolated from atherosclerotic aortas simply by suspending minced tissues in PBS. The characteristics of the calcification process and the enzymatic contents of isolated vesicles were similar to those obtained using collagenase digestion. Correlatively, mineral deposited by calcifiable vesicles isolated from the calcified aortas was also of hydroxyapatite-like phases. Altogether, these observations indicate that (1) aortic calcification is a later event during atherogenesis, (2) calcifiable vesicles are loosely bound to the matrices of the lesions as the result of the disease process and (3) similarities in the mineral phases between those in aortas and by vesicles during atherogenesis further support the role of calcifiable vesicles in dystrophic calcification.

Selective synthesis of bone morphogenetic proteins-1, -3, -4 and bone sialoprotein may be important for osteoinduction by Saos-2 cells.

An ability to induce new bone formation at a required site would represent a considerable advance in bone repair and tissue engineering. It has been shown that the healing of critical-size bone defects in rats can be augmented by extracts of Saos-2 cells. These human osteosarcoma cells uniquely contain a bone-inducing activity, whereas other human osteosarcoma cells, e.g., U-2 OS cells, cannot replicate the osteoinductive capacity. To understand the necessary components of the Saos-2 bone-inducing activity, this study compared osteoinductive Saos-2 cells with non-osteoinductive U-2 OS cells with respect to the synthesis of bone morphogenetic proteins (BMPs)-1, -2, -3, -4, -5, -6, and -7 and the non-collagenous matrix proteins bone sialoprotein (BSP), osteonectin (ON), osteopontin (OPN), and osteocalcin (OC). The main differences were abundant synthesis of BMP-1/tolloid, BMP-3, -4, and BSP by Saos-2 cells, but absence or reduced synthesis in U-2 OS cells. BMP-2 and -7 were present in low amounts in both cell types, while BMP-5 and -6 were more abundant in U-2 OS cells, suggesting that these BMPs were of lesser importance for the osteoinductivity of Saos-2 cells. However, a relatively high expression of BMP-3 and -4, together with BMP-1/tolloid, may be important for the osteoinductive capacity of Saos-2 cells. The inability of U2-OS cells to induce bone, despite expressing most of the BMPs, may be due to an insufficiency of tolloid, BMP-3 or -4, BSP, and/or other unknown factors. A better understanding of the necessary components of the Saos-2 cell bone-inducing agent may, in future, lead to clinically useful Saos-2 cell products for bone repair and tissue engineering.

Mercury: results on mass, radius, ionosphere, and atmosphere from mariner 10 dual-frequency radio signals.

Analysis of the radio-tracking data from Mariner 10 yields 6,023,600 +/- 600 for the ratio of the mass of the sun to that of Mercury, in very good agreement with values determined earlier from radar data alone. Occultation measurements yielded values for the radius of Mercury of 2440 +/- 2 and 2438 +/- 2 kilometers at laditudes of 2 degrees N and 68 degrees N, respectively, again in close agreement with the average equatorial radius of 2439 +/- 1 kilometers determined from radar data. The mean density of 5.44 grams per cubic centimeter deduced for Mercury from Mariner 10 data thus virtually coincides with the prior determination. No evidence of either an ionosphere or an atmosphere was found, with the data yielding upper bounds on the electron density of about 1500 and 4000 electrons per cubic centimeter on the dayside and nightside, respectively, and an inferred upper bound on the surface pressure of 10(-8) millibar.

Venus: mass, gravity field, atmosphere, and ionosphere as measured by the mariner 10 dual-frequency radio system.

Analysis of the Doppler tracking data near encounter yields a value for the ratio of the mass of the sun to that of Venus of 408,523.9 +/- 1.2, which is in good agreement with prior determinations based on data from Mariner 2 and Mariner 5. Preliminary analysis indicates that the magnitudes of the fractional differences in the principal moments of inertia of Venus are no larger than 10(-4), given that the effects of gravity-field harmonics higher than the second are negligible. Additional analysis is needed to determine the influence of the higher order harmonics on this bound. Four distinct temperature inversions exist at altitudes of 56, 58, 61, and 63 kilometers. The X-band signal was much more rapidly attenuated than the S-band signal and disappeared completely at 52-kilometer altitude. The nightside ionosphere consists of two layers having a peak density of 10(4) electrons per cubic centimeter at altitudes of 140 and 120 kilometers. The dayside ionosphere has a peak density of 3 X 10(5) electrons per cubic centimeter at an altitude of 145 kilometers. The electron number density observed at higher altitudes was ten times less than that observed by Mariner 5, and no strong evidence for a well-defined plasmapause was found.

Bistatic-radar detection of lunar scattering centers with lunar orbiter I.

Continuous-wave signals transmitted from Lunar Orbiter I have been received on Earth after they have been reflected from the surface of the moon. The frequency spectrum of the reflected signals is used to locate discrete, heterogeneous, scattering centers on the lunar surface. The scattering centers are probably distinguished from the surrounding terrain by a higher surface reflectivity. Continuous-wave bistatic radar could provide an important new method for the study and mapping of planetary surfaces.