In vitro assessment of the DNA damage and senility of CD117+ stem cells collected from diabetic mice.

OBJECTIVE: Angiogenesis impairment is a common feature of diabetes mellitus (DM), whereas CD117+ bone marrow cells (BMCs) injury might be responsible for such complication. In this study, we studied the effect of hyperglycemia on the DNA damage and senility of CD117+ bone marrow cells. MATERIALS AND METHODS: We isolated CD117+ BMCs from the Streptozotocin (STZ) induced diabetes and healthy control mice. Oxidative stress was detected by flow cytometric analysis. γ-H2AX, which is the DNA damage mark, was detected by using Western blotting and immunofluorescence histochemistry. We also detected the expression of γ-H2AX and p16 by using Western blotting. RESULTS: Compared with the control mice, the level of reactive oxygen species (ROS) was increased significantly in the CD117+ BMCs collected from the diabetic mice (p<0.05), and the percentage of γ-H2AX positive cells was higher significantly (p<0.01). The expression of γ-H2AX and p16 was increased significantly in the CD117+ BMCs from the diabetic mice. CONCLUSIONS: Our experiments demonstrated the oxidative stress in CD117+ BMCs under DM conditions, while accelerating the DNA damage and senility in CD117+ BMCs as well.

Empirical electro-optical and x-ray performance evaluation of CMOS active pixels sensor for low dose, high resolution x-ray medical imaging.

Monolithic complementary metal oxide semiconductor (CMOS) active pixel sensors with high performance have gained attention in the last few years in many scientific and space applications. In order to evaluate the increasing capabilities of this technology, in particular where low dose high resolution x-ray medical imaging is required, critical electro-optical and physical x-ray performance evaluation was determined. The electro-optical performance includes read noise, full well capacity, interacting quantum efficiency, and pixels cross talk. The x-ray performance, including x-ray sensitivity, modulation transfer function, noise power spectrum, and detection quantum efficiency, has been evaluated in the mammographic energy range. The sensor is a 525 x 525 standard three transistor CMOS active pixel sensor array with more than 75% fill factor and 25 x 25 microm pixel pitch. Reading at 10 f/s, it is found that the sensor has 114 electrons total additive noise, 10(5) electrons full well capacity with shot noise limited operation, and 34% interacting quantum efficiency at 530 nm. Two different structured CsI:Tl phosphors with thickness 95 and 115 microm, respectively, have been optically coupled via a fiber optic plate to the array resulting in two different system configurations. The sensitivity of the two different system configurations was 43 and 47 electrons per x-ray incident on the sensor. The MTF at 10% of the two different system configurations was 9.5 and 9 cycles/mm with detective quantum efficiency of 0.45 and 0.48, respectively, close to zero frequency at approximately 0.44 microC/kg (1.72 mR) detector entrance exposure. The detector was quantum limited at low spatial frequencies and its performance was comparable with high resolution a: Si and charge coupled device based x-ray imagers. The detector also demonstrates almost an order of magnitude lower noise than active matrix flat panel imagers. The results suggest that CMOS active pixel sensors when coupled to structured CsI:Tl can be used for conventional and advanced digital mammography due to their low noise, high resolution performance.

Role of A(2A)-adenosine receptor activation for ATP-mediated coronary vasodilation in guinea-pig isolated heart.

1. Adenosine-5'-triphosphate (ATP) and adenosine are potent coronary vasodilators. ATP is rapidly converted to adenosine by ectonucleotidases. We examined whether coronary vasodilation caused by exogenous ATP is mediated by P(2) receptor activation or by A(2A)-adenosine receptor activation. 2. Effects of interventions on coronary conductance were determined by measuring coronary perfusion pressure in guinea-pig isolated hearts perfused at a constant flow of 10 ml min(-1). 3. ATP and adenosine both caused sustained, concentration-dependent increases of coronary conductance. Maximal responses to both agonists were equivalent. The values of pD(2) (+/-s.e.mean) for ATP and adenosine were 6.68+/-0.04 and 7.06+/-0.05, respectively. Adenosine was significantly more potent than ATP (P<0. 0001, n=10). 4. The values of pIC(50) for the selective A(2A)-adenosine receptor antagonist SCH58261 to antagonize equivalent responses to ATP and adenosine were 8.28+/-0.08 and 8.28+/-0.06 (P=0.99, n=6), respectively. 5. The non-selective adenosine receptor antagonists xanthine amine congener (XAC) and CGS15943 antagonized similarly the equivalent vasodilations caused by ATP (pIC(50) values 7.48+/-0.04 and 7.45+/-0.06, respectively) and adenosine (pIC(50) values 7. 37+/-0.13 and 7.56+/-0.11). 6. In contrast to ATP and adenosine, the two P(2) agonists 2-methylthio-ATP and uridine-5'-triphosphate failed to cause stable increases of coronary conductance, caused desensitization of vasodilator responses, and were not antagonized by SCH 58261, 8-parasulphophenyltheophylline, or XAC. 7. Glibenclamide attenuated coronary vasodilations caused by ATP and adenosine by 88 and 89%, respectively, but failed to attenuate those caused by 2-methylthio-ATP. 8. These results strongly suggest that sustained, submaximal coronary vasodilation caused by exogenous ATP is entirely mediated by adenosine acting upon A(2A)-adenosine receptors.

Inflammatory agents regulate in vivo expression of fractalkine in endothelial cells of the rat heart.

Fractalkine is distinguished structurally from other chemokines in that it contains a mucin-like stalk that tethers a CX3C chemokine module to a transmembrane-spanning region; its expression in cultured endothelial cells has been shown to be up-regulated by tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1). The purpose of this study was to determine whether fractalkine is expressed, in a proinflammatory agent-regulated manner, by cardiac endothelial cells in vivo. Steady state levels of fractalkine mRNA were increased in rat cardiac tissues after in vivo treatment with lipopolysaccharide (LPS), IL-1, or TNF-alpha. In situ hybridization and immunohistochemical analysis revealed that endothelial cells of the coronary vasculature and endocardium were the principal source of proinflammatory agent-inducible fractalkine, although some fractalkine immunoreactivity was also found on the myocytes. These data are the first demonstration of in vivo cardiac endothelial cell fractalkine expression and regulation by proinflammatory agents such as LPS, IL-1, or TNF-alpha. Cardiac endothelial cell-expressed fractalkine may contribute to the influx of leukocytes into the heart during inflammation.

A comparison of an A1 adenosine receptor agonist (CVT-510) with diltiazem for slowing of AV nodal conduction in guinea-pig.

1. The purpose of this study was to compare the pharmacological properties (i.e. the AV nodal depressant, vasodilator, and inotropic effects) of two AV nodal blocking agents belonging to different drug classes; a novel A1 adenosine receptor (A1 receptor) agonist, N-(3(R)-tetrahydrofuranyl)-6-aminopurine riboside (CVT-510), and the prototypical calcium channel blocker diltiazem. 2. In the atrial-paced isolated heart, CVT-510 was approximately 5 fold more potent to prolong the stimulus-to-His bundle (S-H interval), a measure of slowing AV nodal conduction (EC50 = 41 nM) than to increase coronary conductance (EC50 = 200 nM). At concentrations of CVT-510 (40 nM) and diltiazem (1 microM) that caused equal prolongation of S-H interval (approximately 10 ms), diltiazem, but not CVT-510, significantly reduced left ventricular developed pressure (LVP) and markedly increased coronary conductance. CVT-510 shortened atrial (EC50 = 73 nM) but not the ventricular monophasic action potentials (MAP). 3. In atrial-paced anaesthetized guinea-pigs, intravenous infusions of CVT-510 and diltiazem caused nearly equal prolongations of P-R interval. However, diltiazem, but not CVT-510, significantly reduced mean arterial blood pressure. 4. Both CVT-510 and diltiazem prolonged S-H interval, i.e., slowed AV nodal conduction. However, the A1 receptor-selective agonist CVT-510 did so without causing the negative inotropic, vasodilator, and hypotensive effects associated with diltiazem. Because CVT-510 did not affect the ventricular action potential, it is unlikely that this agonist will have a proarrythmic action in ventricular myocardium.

MMP-8 is the predominant collagenase in healing wounds and nonhealing ulcers.

BACKGROUND: The initial cleavage of collagen by collagenase represents the rate-limiting step in the degradation of this central extracellular matrix protein. Chronic nonhealing ulcers, especially pressure ulcers, typically contain elevated levels of collagenolytic activity. However, there have been no detailed attempts to identify the source of these collagenases and their activity either in normal healing wounds or in chronic nonhealing ulcers. MATERIALS AND METHODS: Levels of the matrix metalloproteinases, MMP-1 and MMP-8, and the tissue inhibitor of matrix metalloproteinases, TIMP-1, were measured in fluids and tissues of healing human wounds and nonhealing ulcers by ELISA. Relative MMP-1 and MMP-8 levels were also analyzed by substrate preference in a functional assay. RESULTS: The patterns of the collagenases MMP-1 and MMP-8 in healing wounds were distinct, with MMP-8 appearing in significantly greater amounts than MMP-1. Chronic nonhealing ulcers were characterized by significantly higher levels of MMP-1 and MMP-8, and lower levels of TIMP-1, than in healing wounds. Levels of both MMP-1 and MMP-8 varied greatly in chronic ulcers, although MMP-8 was always the predominant collagenase present in these wounds. Interestingly, these collagenases were present almost exclusively in their inactive forms in healing wounds, whereas nonhealing ulcers possessed significant levels of the active forms of these enzymes. CONCLUSIONS: These results clearly demonstrate that the neutrophil-derived MMP-8 is the predominant collagenase present in normal healing wounds and suggest that overexpression and activation of this collagenase may be involved in the pathogenesis of nonhealing chronic ulcers. In addition, excessive collagenolytic activity in chronic ulcers is made possible, partly because of the reduced levels of the inhibitor, TIMP-1.

Dynamics of the matrix metalloproteinases MMP-1 and MMP-8 in acute open human dermal wounds.

Extracellular matrix degradation during dermal wound healing involves multiple levels of regulation by several enzymes of the matrix metalloproteinase family, their activators, and their inhibitors. This study tested the hypothesis that a temporal pattern of interstitial collagenase appearance occurs during normal dermal wound healing, with matrix metalloproteinase-8 originating from neutrophils appearing earlier than the fibroblast-derived matrix metalloproteinase-1. Open (6 mm) full-thickness dermal wounds, which were covered by transparent occlusive dressings, were made in healthy human volunteers (n = 20). Wound fluids from under the dressings were collected daily through day 8, and wound tissue biopsies were obtained on days 0, 2, 4, 14, and 28. Collagenases were extracted from homogenized tissue biopsies for analysis. Samples were analyzed for the presence of matrix metalloproteinase-1 and matrix metalloproteinase-8 by enzyme-linked immunosorbent assays and by collagenase activity assays using purified types I and III collagen as substrates. In addition, tissue inhibitor of metalloproteinases-1 and matrix metalloproteinase-1/tissue inhibitor of metalloproteinases-1 complexes in wound fluids were measured. Results showed a differential temporal pattern of matrix metalloproteinase-1 and matrix metalloproteinase-8 in wound exudates with peak levels of matrix metalloproteinase-8 occurring on day 4 and matrix metalloproteinase-1 peak levels on day 7. Maximal levels in tissue for both enzymes occurred on day 2. At all time points examined, levels of matrix metalloproteinase-8 were statistically higher than matrix metalloproteinase-1 (100-fold to 200-fold). Tissue inhibitor of metalloproteinases-1 levels declined over time, whereas levels of matrix metalloproteinase-1/tissue inhibitor of metalloproteinase-1 complexes increased to a plateau on day 7. This study provides new evidence implicating matrix metalloproteinase-8 as a major collagenase in healing human dermal wounds. It also shows a temporal pattern in the appearance of the matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, and matrix metalloproteinase-1/tissue inhibitor of metalloproteinases-1 complexes, suggesting that a tightly regulated pattern of expression of matrix metalloproteinases and their inhibitors is essential for normal wound healing in humans.

Wound fluids from human pressure ulcers contain elevated matrix metalloproteinase levels and activity compared to surgical wound fluids.

Fluid from acute surgical wounds and from nonhealing pressure ulcers was examined for the presence of several matrix metalloproteinases. Gelatin zymography demonstrated the presence of two major gelatinases with apparent molecular masses of 72 kDa and 92 kDa and two minor gelatinases with apparent mobilities of 68 kDa and 125 kDa. Antigen-specific sera identified the 72-kDa protein as matrix melloproteinase-2. The same sera also reacted with the 68-kDa protein, which is consistent with it being an activated form of matrix metalloproteinase-2. Antigen-specific sera identified the 92-kDa and 125-kDa proteins as matrix metalloproteinase-9. Levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 were elevated more than 10-fold and 25-fold, respectively, in fluids from pressure ulcers compared with fluids from healing wounds. Examination of total potential and actual collagenolytic activity revealed that fluid from pressure ulcers contained significantly greater levels of both total and active collagenase compared with that of acute surgical wounds. In addition, an enzyme-linked immunosorbent assay demonstrated that fluids from pressure ulcers contained significantly more collagenase complexed with the inhibitor, tissue inhibitor of metalloproteinases. Together, these observations suggest that an imbalance exists between levels of matrix metalloproteinases and their inhibitors in the fluids of pressure ulcers and that this is primarily the result of elevated levels of the matrix metalloproteinases. The presence of excessive levels of activated forms of matrix-degrading enzymes at the wound surface of pressure ulcers may impede the healing of these wounds and may be relevant to the development of new rationales for treatment.

Transforming growth factor-beta1 reduces expansion of open wounds in the fetal rabbit.

Open wounds in the fetal rabbit do not heal by contraction and actually expand between 60% and 90% over a period of 5 days. Experiments were carried out to determine whether transforming growth factor-beta1 can reduce expansion of open wounds in the fetal rabbit. This study was based on the concept that transforming growth factor-beta1 causes differentiation of fibroblasts into contractile fibroblasts or "myofibroblasts." To test this hypothesis, pregnant New Zealand White rabbits underwent laparotomy and hysterotomy on day 24 of gestation. A circular full-thickness cutaneous wound was made on the back of each fetus. After wounding, either vehicle alone or vehicle with transforming growth factor-beta1 was applied topically to the wound site, and each fetus was then returned to the uterus. The hysterotomy and laparotomy were closed in standard fashion. On postoperative day 5, fetuses were harvested by repeat Cesarean section. Wound areas were determined from photographs, calculated as percentage of original wound size, and expressed in square millimeters. In addition, a portion of each wound was fixed and processed for histologic and immunohistochemical analysis. At harvest, the control wounds had expanded by an average of 87% of the original area. In marked contrast, the transforming growth factor-beta1-treated wounds had only expanded an average of 16%. Thus, transforming growth factor-beta1 significantly decreased the area of the open fetal wounds compared with control (p < 0.001). By histologic examination, no significant difference was found between the test group and the control group with regards to inflammation, neovascularization, collagen deposition, elastin content, glycosaminoglycan content, or hyaluronic acid content. Most notably, however, there was an increased density of fibroblasts in the transforming growth factor-beta1-treated group. In addition, immunohistochemical staining with an anti-alpha-smooth muscle actin antibody showed the presence of contractile fibroblasts in the wound margins in the transforming growth factor-beta1-treated group but failed to show any positive-staining fibroblasts in the matrices of the control group. These results indicate that open wounds in the fetal rabbit treated in vivo with transforming growth factor-beta1 were significantly smaller than control wounds. This process appears to result from the recruitment and differentiation of normal dermal fibroblasts into contractile fibroblasts containing alpha-smooth muscle actin.

Frequency response of the Fourcin electroglottograph and measurement of temporal aspects of laryngeal movement during swallowing.

The purpose of this investigation was to study the frequency response of a modified Fourcin EGG at frequencies associated with the slow varying laryngeal movement of swallowing and to compare those findings with the response characteristics of the EGG at frequencies associated with phonation. Frequency-dependent differences were found. At very low frequencies the EGG output was found to be the derivative of the changes in neck impedance; at higher frequencies the EGG output directly represented the changes in neck impedance. Three of the four phases of the pharyngeal stage of the swallow are represented by the EGG signal.

[Use of the Powell method to optimize the composition of HPLC mobile phase--the separation of ephedrine and pseudoephedrine].

The isomers ephedrine and pseudoephedrine are difficult to be separated by HPLC. In this paper, the Powell method was successfully introduced to optimize the composition of mobile phase in HPLC. On a mu Bondapak C18 column (8-10 microns, 3.9 mm x 30 cm), KH2PO4 solution methanol mobile phase system was optimized for separating ephedrine and pseudoephedrine by the Powell method. The optimized composition of the mobile phase was found as: 0.01 mol/L KH2PO4 -MeOH = 94: 6 (V/V). The Powell method was carried out in a process illustrated by tables and figures and 0.95 peak separation function was achieved.

[Determination of ephedrine, pseudoephedrine and strychnine in jiufen san by HPLC].

An HPLC method was established to separate and determine ephedrine (I), pseudoephedrine (II) and strychnine (III) in Chinese traditional medicine, Jiufen San, on a mu-Bondapak C18 column (10 microns, 3.9 mm x 30 cm) by using 0.01 mol/L KH2PO4-methanol as mobile phase. The programmed gradient elution was carried out and recoveries were determined as 98.94 +/- 2.2% for I, 97.37 +/- 1.9% for II and 100.7 +/- 1.9% for III respectively. An extracting and pretreatment method was designed and the result showed that the extracting efficiency of this method was 1.33 times (I) and 1.29 times (II) higher than those of the Chinese Pharmacopoeia method.