Analysis And Synthesis Of Mechanisms Pdf

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Kinematic Analysis and Synthesis of Mechanisms

Cardiovascular disease is the leading cause of morbidity and mortality in postmenopausal women. EM acolbifene is a fourth-generation selective ER modulator SERM exerting complete antiestrogenic effects on the breast and uterus.

EM potently inhibits bone resorption and induces positive lipid modifications in estrogen-deficient animals. As most of the cardioprotective actions of estrogen are exerted directly at the vascular level, we studied the effects of EM on endothelial production of nitric oxide NO in vitro and in vivo.

Upon pharmacological comparison, EM is markedly more potent than the SERMs raloxifene and tamoxifen in increasing NO synthesis from endothelial cells. In ovariectomized and fertile rats, EM increases aortic eNOS expression and enzymatic activity at low, but not at higher, dosages.

The present data show that EM acolbifene has estrogen-like activity on the vascular wall, directly increasing NO production through genomic and nongenomic mechanisms in vitro and in vivo. The endocrine modifications of the climacteric exert detrimental effects on the blood vessels, which add to the ageing process 1.

Indeed, menopause has recently been advocated as a new independent cardiovascular risk factor in women 2 , 3. Hormone replacement therapies HRTs have been shown to be associated with improved cardiovascular risk parameters 4 , fewer cardiovascular events 5 , and reduced cardiovascular mortality 6.

However, this has been questioned by two large trials that provided evidence that HRT in elder women with established coronary disease has no beneficial effect on future cardiovascular events or survival 7 , 8 as well as on atherosclerotic lesions progression 9 , therefore raising an ongoing debate about the usefulness of these treatments for cardiovascular disease prevention 3 , Although estrogen has traditionally been considered to exert cardioprotective actions through positive modifications of the lipid profile 4 , there has been accumulating evidence supporting the concept that the major part of the beneficial effects of female sex hormones are exerted directly at the vascular level through lipid-independent mechanisms 11 , Furthermore, estrogen is able to recruit and activate both transcriptional genomic signaling mechanisms as well as nontranscriptional or nongenomic pathways in vascular cells, thus regulating physiologically important functions such as nitric oxide NO synthesis by endothelial cells 13 — Selective ER modulators SERMs are molecules exerting estrogen agonistic or antagonistic activities in different tissues EM acolbifene is a fourth-generation SERM characterized by complete antiestrogenic effects on the mammary gland and endometrium while being a potent inhibitor of bone resorption in animal models This unique endocrine profile of EM heralds the possibility that this compound could be an effective tool for postmenopausal women However, an ideal compound for HRT should mimic the effects of natural estrogen on the cardiovascular system.

In this regard, it is known that the various SERMs induce different and not always beneficial modifications of the lipid profile in postmenopausal women 16 , and we have shown that parent SERMs are not equally effective in exerting antiatherogenic effects on human endothelial cells Although preliminary data indicate that EM effectively corrects the lipid modifications associated with ovariectomy in rats 19 , 20 , no data on possible direct actions on the vessel wall have been described to date.

As endothelial cell-derived NO plays a critical role in cardiovascular physiology and disease, we studied whether EM acolbifene has direct vascular effects in vitro on human endothelial cells as well as in vivo in fertile or ovariectomized female rats. All experiments were performed on confluent monolayers of endothelial cells. Fertile female Wistar rats, weighing — g, were purchased from Harlan Nossan Italy.

After 14 d from arrival, some rats were ovariectomized during the same estrous cycle stage as indicated by daily vaginal smears. After complete healing of the surgical wounds, ovariectomized rats as well as fertile animals were started on treatment with EM HCl or E2 valerate or both for 14 d. HCl was administered orally to all animals as a 0. All fertile rats were in the same stage of the estrous cycle at the beginning of treatment. Rat aortas were homogenized in the same buffer, and after centrifugation to discard the cell debris, the supernatants were used for eNOS assay.

Louis, MO as previously described Extracts incubated with the eNOS inhibitor, N G -nitro- l -arginine methyl ester 1 m m , served as the blank. Converted eNOS activity was obtained by subtracting the blank from the samples. The counts per min value of untreated samples was taken as 1, and all other values were normalized to untreated samples and expressed as the fold increase vs.

NO production by endothelial cells was determined by a modified nitrite assay using 2,3-diaminonaphtalene as described previously The fluorescence of 1- H -naphtotriazole was measured with excitation and emission wavelengths of and nm, respectively.

Standard curves were constructed with known amounts of sodium nitrite. Specific antibodies vs. N , Akt catalog no. SC , PR catalog no. SC , and AR catalog no. The blots were hybridized with secondary antibodies coupled to horseradish peroxidase as previously described Immunodetection was accomplished using enhanced chemiluminescence.

Estrogen-deprived HUVEC treated with increasing concentrations of EM for 30 min released NO measured as the stable metabolites nitrites in the culture medium in a concentration-dependent fashion Fig. EM increases NO release through nongenomic mechanisms. Both inhibitors were added 30 min before initiating treatment with EM The blots in C are representative of 3 different experiments, with comparable results. As 30 min was possibly too short a time for a genomic effect, we checked whether EM triggered an enzymatic activation of eNOS, independently from eNOS gene regulation.

As shown in Fig. As further proof, we checked for eNOS protein amount in endothelial cells treated with EM for 30 min, and no differences were found Fig. As a loading control, the membrane was reblotted for the endothelial marker vWF, which is not induced or down-regulated under the investigated conditions Fig. Furthermore, we treated endothelial cells with the SERM in the presence or absence of the transcriptional inhibitor actinomycin D or the translational inhibitor cycloheximide.

Under such conditions no inhibition of EMinduced NO release by the RNA or protein synthesis blockers was observed, thus confirming that rapid activation of eNOS by EM is exerted through nontranscriptional and nontranslational mechanisms Fig.

An initial 2- to 2. The same kinetic profile was seen for eNOS activation in cell lysates Fig. Characterization of the rapid NO synthesis induction by EM The blots are representative of 3 different experiments with comparable results. P, Phosphorylated. The ultra-rapid 5—10 min increase in NO synthesis Fig.

As upon activation of PI3K by liganded ER the downstream effector Akt was activated in endothelial cells 13 , we tested whether the same ensued upon treatment with EM Akt phosphorylation on both residues was prevented by pretreatment with ICI , Fig. A, Increasing concentrations of EM, raloxifene or tamoxifen were used to treat estrogen-deprived, serum-starved endothelial cells for 30 min, and nitrite concentrations in the culture medium were measured.

E2 alone. Nitrite measurements showed a synergistic effect of EM addition Fig. As illustrated in Fig. The increase in eNOS by EM was concentration dependent, but a significant induction was found even at the lowest concentration used 0.

The induction of eNOS expression in endothelial cells was not a nonspecific effect of EM, because under the same experimental conditions there was no increase in the endothelial marker vWF expression Fig.

A, Estrogen-deprived endothelial cells were treated with EM 1 n m for 48 h, and nitrite release was assayed in cell culture medium. Cell lysates were immunoblotted with antibodies vs. C, Increasing concentrations of EM, raloxifene, or tamoxifen were used to treat endothelial cells for 48 h, and nitrite concentrations in the culture medium were measured. Upon comparison with raloxifene and tamoxifen, EM showed a high potency, similar to the rapid, nongenomic effects described above.

Indeed, EM was markedly more potent than the other SERMs and showed a bell-shaped curve of efficacy, with a decreasing stimulatory effect at the highest concentration tested, whereas raloxifene and tamoxifen exerted stimulatory effects at much higher concentrations Fig.

To test whether the findings of the effects of EM on endothelial cells are also observed in vivo , we treated ovariectomized or fertile female Wistar rats for 14 d, and eNOS protein content and enzymatic activity in abdominal aortas were assayed. Ovariectomized rats have markedly lower amounts of eNOS in aortic tissues compared with fertile animals Fig. The loss of eNOS after castration is dependent on estrogen deficiency, as it can be reversed by oral administration of E2 valerate in a dose-dependent fashion Fig.

No changes were seen under the same conditions for vWF expression Fig. In vivo aortic eNOS expression is estrogen dependent. Ovariectomized Wistar rats were treated orally for 14 d with vehicle or different dosages of E2 valerate, and abdominal aorta tissue extracts were assayed for eNOS and vWF protein contents. A total of 12 animals for each experimental condition were assayed with consistent results, and representative blots are shown. Each band in A and C represents the aortic protein extract from a different animal.

EM administration restored aortic expression of eNOS in ovariectomized animals, with maximal induction observed with the 0. Enzymatic eNOS activity in the aortic homogenates followed a similar pattern, with a significant increase at the low dose and lower efficacy at higher doses, although the decrease at high doses was not statistically significant Fig.

To check for the possible antiestrogenic effects suggested by in vitro experiments, we administered to ovariectomized rats either E2 valerate alone or together with different doses of EM and were able to demonstrate the same phenomenon observed in vitro , with a synergic action of EM at the lower dose, where it further potentiates E2-dependent eNOS induction, but the loss of such effect at the higher doses Fig.

This supports the possibility of an optimal dose for EM, beyond which the pharmacological efficacy on endothelial eNOS expression decreases. Ovariectomized Wistar rats were treated orally for 14 d with vehicle or with E2 valerate alone or together with different doses of EM, and aortic extracts were assayed for eNOS and vWF protein contents.

Each band represents the protein extract from a different aorta. Twelve animals for each experimental condition were assayed with consistent results, and representative blots are shown. Cardiovascular disease is a major concern for postmenopausal women, and estrogen deficiency represents an important concurrent factor in the pathophysiology of vascular degeneration. HRT is beneficial to postmenopausal women, but the side-effects may be significant, and the fear of developing uterine and breast cancer is an important reason for noncompliance.

The development of compounds that act as estrogens selectively on the desired tissues, whereas behaving as estrogen antagonists on the mammary gland and endometrium thus has major clinical significance, but one of the required goals for an ideal molecule is that the positive effects of estrogens on the cardiovascular system not be lost.

Estrogen actions on the cardiovascular system are multifactorial, but the main part of the beneficial effects is exerted through actions on vascular cells 11 , Thus, ideally, a proposed cardiovascular-active SERM should be able to exert positive effects on the vascular wall Our current knowledge of the vascular effects of SERMs is still very limited 12 ; however, there are indications that not all SERMs have the same effects at this level NO synthesis by endothelial cells is of paramount importance for the regulation of vascular tone and blood flow and for control of the hemostatic process Furthermore, endothelium-derived NO is a potent antiinflammatory and antiatherogenic factor, being able to prevent endothelial cell dysfunction and vascular degenerative processes NO is a primary vascular target of estrogens.

In fact, estrogens stimulate the synthesis and release of NO through the stimulation of eNOS gene expression 26 as well as through nontranscriptional activation of eNOS enzymatic activity 13 — This finding is of potential pathophysiological importance, as this SERM has been shown to relax rabbit coronary arteries through a NO-dependent mechanism Our present data show that the fourth-generation SERM, EM acolbifene , which acts as a complete estrogen antagonist in the mammary gland and endometrium, exerts a typical estrogen-like effect on endothelial cells, triggering NO synthesis.

We also demonstrate that EM has a double mechanism of action, inducing a rapid nongenomic stimulation of eNOS activity as well as an up-regulation of eNOS protein upon longer exposure. Nongenomic signaling through ERs is of primary importance for estrogen action.

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TSH stimulation of orotic acid conversion to pyrimidine nucleotides in bovine thyroid slices was investigated. Precursors of phosphorylated ribose, an essential component in the reactions involved, such as glucose, ribose and inosine, also stimulated nucleotide formation. The presence of glucose enhanced the magnitude of the TSH response, whereas both ribose and inosine completely abolished the stimulating effect of the hormone. The potencies of the precursors in stimulating nucleotide synthesis appeared to be inversely related to their expected values as substrates for energy production, with glucose being the poorest stimulant and inosine the most potent. Aspartate, succinate and acetate, which are excellent substrates for thyroid slices, had no influence on nucleotide formation. Neither TSH, glucose nor ribose affected nucleotide synthesis at orotic acid concentrations of 2.

Cardiovascular disease is the leading cause of morbidity and mortality in postmenopausal women. EM acolbifene is a fourth-generation selective ER modulator SERM exerting complete antiestrogenic effects on the breast and uterus. EM potently inhibits bone resorption and induces positive lipid modifications in estrogen-deficient animals. As most of the cardioprotective actions of estrogen are exerted directly at the vascular level, we studied the effects of EM on endothelial production of nitric oxide NO in vitro and in vivo. Upon pharmacological comparison, EM is markedly more potent than the SERMs raloxifene and tamoxifen in increasing NO synthesis from endothelial cells.


This text is an introduction to the analysis and synthesis of mechanisms and machines, with an emphasis on the first. The intended audience is undergraduates.


Kinematic Analysis and Synthesis of Mechanisms

Lehigh Preserve has a new look! Please contact us at preserve lehigh. Thank you for your patience. Reuther, Dana. Mechanical Engineering.

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This work was supported by grants from the Canadian Arthritis Society C.

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