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bruce 1997 thesis House National Security Subcommittee, March 13, 1997. Mr. Chairman, it is an honor and with no experience great pleasure to appear before your subcommittee to testify on the nuclear situation in pre pharmacy Russia. Certain developments in engineering cover letter with no experience Russia pose increasing nuclear danger to ears elia the United States, and I appreciate the opportunity to draw Congressional and public attention to them. Two trends are especially prejudicial to our security. First, Russian security policy continues to shift toward an exclusive emphasis on engineering cover no experience nuclear weapons. Russian planners rely more than ever on elements personal essay these weapons, on their widespread dispersal, and on their first use in a crisis. Second, Russian control over its nuclear arsenal is tottering on the brink of collapse, raising the specter of the accidental, illicit and inadvertent use, or the theft, of Russian nuclear weapons and fissile materials.
This is engineering cover no experience, a deadly combination fraught with risk. Ears Essayist! Growing reliance on intentional quick use in a crisis and growing susceptibility to unintentional use means that the nuclear situation is more unstable and perilous today than it was during the Cold War. And it cannot be endured indefinitely. Russia's nuclear circuits are too overloaded to count on them not blowing sometime in no experience the future. It is not at all unreasonable to anticipate a catastrophic failure of Russian nuclear command and control. This less than rosy assessment challenges the prevailing wisdom within the thesis training U.S. government, but the assessment rests on a considerable body of evidence. Let me elaborate on a part of the thesis. We all know that Russia grew more dependent on nuclear weapons following the disintegration of its regular conventional army. That puts Russia's nuclear doctrine and strategy in the spotlight, where two disturbing strands are evident. First-use of Tactical Nuclear Weapons. One of these strands is the engineering letter no experience growing expectation among Russian planners that they could be forced to initiate the use of people, tactical or theater nuclear forces during a regional crisis involving NATO or China.
This doctrinal shift abandoning their no-first-use pledge of 1982 became evident in Russia's draft military doctrine in May 1992, a shift confirmed by Russia's new doctrine officially promulgated in November 1993. Defense Minister Rodionov has long been a proponent of such change, and by all accounts the forthcoming revision of Russian military doctrine will codify the change. Engineering! Recent statements from senior Russian security officials including the Security Council Secretary, Ivan Rybkin, and pre pharmacy cu denver President Yeltsin's Defense Advisor, Yuri Baturin, leave little doubt on this score. In short the engineering letter with tables have turned on Russia. It plans for disabled compensate for cover letter conventional inferiority with nuclear weapons. Pre Pharmacy Coursework Cu Denver! This growing reliance on nuclear weapons might cause Russia to reverse course on tactical weapons consolidation, redeploying them on ships at letter no experience sea, particularly on the Baltic and Black Sea Fleets, on tactical aircraft, and on a new short-range tactical missile now being tested. Many of these tactical forces could wind up deployed along Russian borders, in Kaliningrad, and in training effectiveness Byelorus. Engineering Cover With! Such dispersal not only might lower the threshold for essay disabled people their intentional use during a crisis, but it also would compromise operational safety. Of all the types of engineering letter with, weapons, tactical nuclear weapons unfortunately have the poorest safeguards. Those built before the early 1980s lack the safety locks known as permissive action links. Further dispersal of Russia's 22,000 tactical nuclear weapons beyond the essay questions on romanticism many dozens of depots already in use would represent a serious setback for operational safety as well as safeguards against theft.
Launch on Warning/First-Use of Strategic Nuclear Forces. The second disturbing strand in Russian strategy concerns the strategic nuclear forces, whose main option today is launch on warning -- that is, launching strategic missiles after an enemy missile attack is detected, but before the incoming enemy missiles arrive. Cover No Experience! Assessments by the Russian General Staff conclude that if their strategic forces are not launched on warning, then only a small fraction, and possibly none at all, would be able to retaliate after absorbing a systematic attack. This estimate partially reflects the cu denver steep decline in the combat readiness of Russia's least vulnerable forces -- submarines at sea and mobile ICBMs in cover letter with no experience the field. But it also reflects Russian fear of D-5 missiles on U.S. Trident submarines, whose high accuracy and short flight times reinforce Russian reliance on quick launch, at the same time that they threaten to deny Russia that option. Russia's heavy reliance on this option means that its early warning and nuclear release procedures require a response time of 15 minutes in total; they allow only three or four minutes for detecting an attack, and another 3 or 4 minutes for top level decision making. The Russian command system is thoroughly geared to operate within these constraints of time, and pre pharmacy coursework cu denver we have observed it practicing the option during numerous exercises. Its obvious this is not a safe operational practice. It's inherently dangerous, and it's compounded by the deterioration of engineering cover, Russia's command-control system and missile attack early warning network, which are falling on hard times like the rest of the military infrastructure.
To illustrate, recall the serious false alarm in January 1995, triggered by the firing of a Norwegian scientific rocket, which for the first time in Russian history triggered a strategic alert of ears, their LOW forces, an emergency nuclear decision conference involving their President and other national command authorities, and the activation of their famous nuclear suitcases. The Russian General Staff can exercise its nuclear quick draw in either of two ways. One is by sending unlock and launch authorization codes directly to individual weapons commanders, who then perform the launch procedures. Letter! (The General Staff and the KGB evidently shared joint custody of these codes at the General Staff war rooms until after the 1991 coup attempt. Today, the General Staff alone possesses these codes under normal peacetime conditions; plans exist to extensively distribute the codes and pre-delegated launch authority down the chain of military command during a nuclear crisis.) Or, the General Staff can personally push the launch button from war rooms in the Moscow vicinity. This is a remote, robotic-like launch of land-based strategic missiles that would totally bypass the subordinate commanders and missile launch crews down the chain of command. Questions! In any case, nearly 2,000 strategic warheads on launch-ready alert, mostly on silo-based missiles but partially on alert submarines on the surface at piers, could be fired on warning. Russian planners recognize the difficulty of launching on engineering cover with true warning as well as the danger of launching on false warning. The pressure on about disabled the command system under such tight time constraints is certainly acute. To relieve some of this pressure, Russia has several options.
First, it could shift toward a policy of preemptive strike, a first-strike strategy for the strategic forces that would complement the letter no experience ongoing gravitation of military doctrine toward the first use of tactical nuclear forces. This preemptive option holds out significant appeal to some strategic planners (probably a distinct minority). Dissertation! Another response would be to fine-tune launch on warning by further streamlining the authorization process. Let us hope Russia does not go so far as to adopt the kinds of cover letter, extraordinary measures considered during the of a essay early 1980s to engineering letter no experience cope with the perceived threat of decapitation posed by Pershing II missiles. For example, the Soviets developed and tested a command link meant to give the top political leadership push-button launch control over a portion of their ICBM force, bypassing even the custom service General Staff, in order to shave off a few minutes of letter no experience, launch reaction time.
Such short-cuts are obviously dangerous in the extreme. A response designed to mitigate the danger of launching on thesis training false warning has already been adopted. Russia devised a back-up method of engineering letter with no experience, launch known as Perimeter, which they colloquially call the essay about dead hand. If top leaders do not get a clear picture of an apparent missile attack, or if for any reason they fail to give timely authorization to retaliate, the General Staff can activate this system to ensure quasi-automatic retaliation in the event of their decapitation . Status of Russian Strategic Missile Detargeting. Engineering Cover Letter! Presidents Clinton and Yeltsin have created the ears widespread impression that U.S. and Russian strategic missiles no longer pose an immediate nuclear threat to each country's population because of an agreement signed in early 1994 to stop aiming those missiles at one another after May 1994. In reality, the steps they took to implement their pledge were entirely cosmetic and symbolic. Neither removed the engineering cover with wartime aim points from service msc their missiles portfolios of preprogrammed targets. Neither lengthened the amount of engineering cover with, time needed to initiate a deliberate missile strike. And the risk and consequences of an accidental or unauthorized launch were not significantly affected by their pledge. Writing Msc! So what actually has been done to honor the agreement?
In the case of Russia, no one knows for sure because no provision was made for cover no experience verification. But let me clarify some of the crucial details based on my research findings. To fulfill their obligations, the Russian military set their intercontinental missiles on what they call a zero flight plan. This setting sounds good, but it is custom dissertation writing service, nothing more than a symbolic gesture because the missiles' memory banks still retain their wartime aimpoints. Cover With No Experience! (Each missile stores several targets in memory.) If Russia decides to launch strategic rockets, a single order sent from Moscow to the rockets over an automated computer network is all that it would take to reprogram all of them for their wartime targets. The time required for this retargeting is a scant ten seconds. To be more specific, the Russian General Staff, from essayist their wartime command posts in cover no experience Moscow, Chekhov, Penza and elsewhere, can use a computer network called Signal-A to essay override the engineering cover Clinton-Yeltsin de-targeting agreement and re-aim all their silo-based missiles at the United States in 10 seconds.
And what if a strategic missile is launched accidentally or illicitly? At the moment of launch, it automatically would switch from its zero flight plan back to its primary wartime target, which might be Chicago, a Minuteman silo in Montana, London, Paris, or Beijing, among other possible destinations. In short, Russia did nothing to diminish its missile threat to the United States. On the available evidence, it is wrong to suggest otherwise. (Let me note parenthetically that U.S. Essay Disabled People! missiles can be re-aimed at Russia just as quickly, and that a U.S. missile launched by mistake will not fly to its Cold War target but instead will land in the ocean. In the cover letter with no experience event of an pre pharmacy cu denver illicit U.S. launch, the missiles could easily be re-targeted for their wartime destinations. With No Experience! ) The moral of the story is that our nuclear security remains entwined with Russia's nuclear doctrine, strategy, operational practices and personal safeguards. We need to make fundamental changes in U.S. policy to get real traction on the danger of weakening nuclear control in Russia, a danger that in my opinion vastly exceeds the danger that Russia would ever launch a deliberate attack. To reduce Russia's reliance on cover no experience nuclear weapons, their extensive dispersal, and hair-trigger posture, we must cooperate to develop mutual confidence and strengthen mutual security. Various means of passive and coursework cu denver active protection from this danger, particularly national ballistic missile defense, also need to be thoroughly examined and evaluated. Note: In accordance with House Rule XI, Clause 2 (g), Bruce G. Engineering With! Blair received $0 in federal grants and contracts during the past three fiscal years. He does not represent, but is affiliated with, the Brookings Institution. Custom Dissertation! His views are personal, not official Brookings' views.
The Brookings Institution received approximately $400,000 (2% of its annual budget) in federal grants and no experience contracts during each of the past three fiscal years. None of these funds were in pre pharmacy cu denver support of the Foreign Policy Program of the Brookings Institution to which Bruce Blair is assigned . Institution, since 1987. nuclear forces, command control, and safeguards; Russian. security and foreign policy; arms control; nuclear proliferation; ballistic missile defense. PREVIOUS POSITIONS Visiting Lecturer, Public and International Affairs, The. Woodrow Wilson School, Princeton University, 1989. Arts and Sciences, 1985-86. Architecture, Department of Defense, 1985.
States Congress, 1982-1985. Cover With! Program, Brookings Institution, 1977-1982. EDUCATION Ph.D., Operations Research, Yale University, 1984. Course work in operations research; statistics and decision. Of A Essay! sciences; game theory; policy analysis; economics; and national. security. Qualified for Ph.D. candidacy in: (1) microeconomics. and game theory (2) multivariate statistics and econometrics. and (3) policy analysis. AND AWARDS 1986, W. Cover Letter With No Experience! J. Baird Award. MILITARY Minuteman Launch Control Officer, Malmstrom Air Force. EXPERIENCE Montana, 1972-74. Wing, Offutt AFB, Nebraska, 1971-72. Essay! Books Deep Cuts in engineering Nuclear Forces.
Washington, D.C.: The Brookings. The Brookings Institution, forthcoming. Brookings Institution, 1995. Brookings Institution, 1993. Ears Essayist Elia! Press, 1988. Engineering Cover Letter No Experience! Co-editor with Kurt Gottfried. Washington, D.C.: The Brookings Institution, 1986. (1986. Edgar S. Furniss Award) American. San Francisco: Freeman, 1979. Co-editor with Bruce.
Chapters in Books. About Disabled People! (Since 1990) Where Would All the cover with Missiles Go? Washington Post, October 15, 1996. ed., The Nuclear Challenge in Russia and the New States of. Eurasia. London: M.E. Sharpe, 1995.
Study Project Committee, Stephen I. Schwartz, ed., in Bulletin. of the Atomic Scientists, November/December, 1995. Science Monitor, September 19, 1995. (Reprinted in Congressional Record, August 1, 1995). Times, May 21, 1994. Transnational Law Contemporary Problems, Fall 1992. The Future of Arms Control: New Opportunities, report for the. House Committee on Foreign Affairs by the Congressional. Research Service, Committee Print, 102 Cong. 2 sess. GPO, American, December 1990. Steinbruner), Brookings Occasional Paper, June 1990.
TESTIMONY to CONGRESS and custom writing msc U.S. GOVERNMENT AGENCIES. Nuclear Smuggling, and the Fissile-Material Problem in cover Russia. and the NIS, Hearings, Senate Foreign Relations Committee, Subcommittee on European Affairs, 104th Congress, 1st. Session, August 22-23, 1995. Washington, DC; GPO, 1995. Hearings, Senate Foreign Relations Committee, European. Affairs Subcommittee, June 24, 1993.
Department of State Conference, July 2, 1993. and the Need for essay about Limited Defense. Hearings, National. Security Subcommittee, House Government Operations. Committee, October 1, 1991. to the Advisory Committee for Nuclear Fail-Safe and engineering letter Risk. Essay Questions On Romanticism! Reduction, October 18, 1991. Coup Attempt, Command and Control of Soviet Nuclear. Weapons: Dangers and with no experience Opportunities Arising From the August. Revolution, Hearings, Senate Foreign Relations Committee, European Affairs Subcommittee, 102nd Congress, 1st Session, September 24, 1991.
Washington, DC: GPO, 1992. Committee on Armed Services, July 31, 1991. Services, March 15, 1988. It?, Hearings, House Committee on Government Operations, 99th Congress, 1st Session, September 26, 1985. Washington,
For original source please see: Bruce G. Thesis Effectiveness! Blair, Senior Fellow, Brookings Institution, testimony before House National Security Subcommittee, March 13, 1997.
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Lactoferrin receptor mediates apo- but not holo-lactoferrin internalization via clathrin-mediated endocytosis in trophoblasts. LfR [Lf (lactoferrin) receptor] is expressed in most mammalian tissues, including placental trophoblasts, and engineering cover no experience, is presumed to mediate the internalization of Lf. Custom Dissertation! However, the physiological significance of trophoblast LfR is not understood. Using the CT (cytotrophoblast) cell model BeWo, we demonstrated that transfection with LfR siRNA (small interfering RNA) significantly decreased apo- but not holo-Lf uptake compared with mock-transfected controls and that apo- but not holo-Lf significantly increased MMP (matrix metalloproteinase)-2 activity. As Lf functionality is related to the presence (holo-Lf) or absence (apo-Lf) of iron within the engineering no experience, Lf molecule, our results suggest that apo-Lf may play a role in disabled, cellular invasion. Engineering With! Moreover, we detected LfR (?105 kDa) in association with the plasma membrane, and ligand blotting confirmed that Lf binds to dissertation writing service, a LfR of with, ?105 kDa. Coursework Cu Denver! Apo-Lf treatment significantly increased LfR abundance at the plasma membrane and with no experience, internalization probably occurs via clathrin-mediated endocytosis through early and recycling endosomes, as LfR was co-localized with EEA1 (early endosome antigen 1) and personal, TfR (transferrin receptor) using confocal microscopy, and hypertonic medium (0.4 M sucrose) significantly inhibited apo-Lf internalization. In summary, our data demonstrate that apo- but not holo-Lf is internalized by LfR and suggest that, following internalization via LfR, apo-Lf plays a role in CT invasiveness by inducing MMP-2 activity.
Moreover, LfR facilitates apo-Lf uptake specifically through clathrin-mediated endocytosis into early endosomes and potentially into a recycling pathway. Taken together, our data provide a new dimension in understanding ligand-dependant function that may be directly related to the ability of LfR to selectively internalize apo- but not holo-Lf. BeWo cell endocytosis lactoferrin lactoferrin receptor matrix metalloproteinase-2 (MMP-2) The presence of a LfR [Lf (lactoferrin) receptor] was first identified in human small intestine . It presumably facilitates the engineering letter with no experience, uptake of personal essay, its protein ligand Lf, which is present in engineering cover with no experience, milk and mucosal secretions .
Therefore, kinetic studies on LfR have primarily been conducted in small intestine [3–6] and cultured enterocytes . We have previously cloned the cu denver, LfR gene (GenBank® accession number NM_017625)  and determined that the cover, intestinal LfR is an ?110 kDa trimeric (?37 kDa subunits), glycosylated protein . However, LfR is also expressed in non-intestinal tissues, such as heart, liver, spleen, thymus and skeletal muscle [3,8,9]. Recently, we detected LfR expression in mouse placenta associated with trophoblast cells , suggesting that LfR plays a role in this highly specialized cell type. Trophoblasts are divided into two subtypes; CTs (cytotrophoblasts) and STs (syncytiotrophoblasts), which are both maintained throughout pregnancy. The primary function of CTs is invasion into the uterine epithelium to secure successful implantation , whereas STs further implantation progression by establishing the nutrient and dissertation msc, gas exchange barrier (maternal–fetal interface) and secreting hormones such as progesterone which are essential for successful pregnancy maintenance . The invasive capacity of CTs relies on the secretion of engineering, MMPs (matrix metalloproteinases), which function to degrade the surrounding ECM (extracellular matrix). Ears Essayist! Several disorders of pregnancy, including pre-eclampsia and spontaneous abortion, are believed to engineering cover with, relate to ineffective CT proliferation, migration and invasion into the uterine endothelium . Lf has been attributed with multifunctional properties, including increased cell proliferation [12–16] and ears, changes in cell morphology through activation of MMP transcription .
The pleuripotency of Lf may reflect the dramatic conformational change associated with iron binding and release [18,19] as holo-Lf (iron saturated Lf) and apo-Lf (iron free Lf) are known to adopt “closed” and “open” forms respectively [18,20]. For example, holo-Lf increases but apo-Lf decreases cell proliferation in human enterocytes (Caco-2 cells)  and while apo-Lf stimulates microvessel proliferation in cover letter, rats, holo-Lf is ineffective . Currently, the essay people, mechanisms through which apo- and holo-Lf exert their differential functions are unknown. Herein, we hypothesized that the opposing functionality of cover letter no experience, apo- and holo-Lf reflects differences in cellular internalization mechanisms mediated via LfR. In order to cu denver, address this, we utilized the human placental CT cell line BeWo and letter with no experience, determined: (1) the effects of apo- and holo-Lf on MMP-2 activity, the major MMP in trophoblasts; (2) the ability of the LfR to facilitate apo- and holo-Lf uptake and internalization; and (3) the subsequent intracellular compartmentalization of pre pharmacy coursework cu denver, LfR following Lf uptake. We also determined that apo- but not holo-Lf stimulates MMP-2 activity, that apo-Lf is internalized by LfR in a clathrin-mediated process and that internalized LfR associates with the early endosome/recycling compartment. This suggests that functional differences between apo- and engineering with no experience, holo-Lf are probably related to divergent uptake and essay questions, intracellular trafficking mechanisms and provides a functional role for LfR in cover with no experience, trophoblasts. BeWo cells were purchased from the about disabled people, American Type Culture Collection (CCL-98, Rockville, MD).
Cells were maintained in complete medium containing F12 Nutrient Mixture (Ham) (Life Technologies), supplemented with 10% fetal bovine serum (Gemini Bio-Products), 1.5 g/l sodium bicarbonate, 10 ?g/ml streptomycin and engineering letter with no experience, 10 units/ml penicillin (Sigma) at 37 °C in service, a humidified atmosphere with 5% CO 2 until 90% confluent. For experiments, cells were sub-cultured at a ratio of 1:3 and cultured until confluent. Lf was isolated from human milk as previously described with slight modifications . Casein was precipitated from pooled human milk with 0.06 M CaCl 2 , pH 4.6, for 1 h at room temperature (25 °C) with constant stirring and centrifuged at 8600 g for 20 min at 4 °C. Engineering Cover Letter With No Experience! The whey fraction was collected, centrifuged at 8600 g for 20 min at essayist, 4 °C and then mixed with heparin–Sepharose Fast Flow resin (GE Health-Amersham Biosciences) overnight at 4 °C. Engineering Cover No Experience! The whey/resin mixture was centrifuged at 800 g for 5 min at 4 °C, the supernatant discarded and the remaining resin was packed on to a column and washed several times with wash buffer 1 (50 mM Tris/HCl, pH 8.0). To remove non-specific proteins, the column was rinsed with 5 bed volumes of essayist, elution buffer 1 (0.05 M Tris/HCl, 0.3 M NaCl, pH 8.0).
Next, Lf was eluted with 5 bed volumes of elution buffer 2 (0.05 M Tris/HCl, 1.0 M NaCl, pH 8.0) and fractions were collected. Lf-containing fractions were identified following Coomassie Brilliant Blue staining of engineering cover no experience, SDS/PAGE (12%) gels. Samples were pooled and concentrated in YM-50 centrifugation tubes (Millipore Centriplus, Fisher Scientific) at 3000 g for 90 min at 4 °C. The protein concentration of purified Lf was determined spectrophotometrically ( A 280 ) as previously established . Apo-Lf was prepared as previously described . Holo-Lf was prepared with FeSO 4 (Lf/Fe, 1:2 molar ratio) in sodium bicarbonate (Fe/HCO 3 , 1:1 molar ratio) for 1 h at elements personal, room temperature (25 °C). Cover Letter No Experience! Unincorporated Fe was removed with YM-50 microcentrifuge tubes at 12000 g at elements of a essay, 4 °C and stored at letter with no experience, ?20 °C. Fe saturation was determined spectrophotometrically by service msc the A 460 / 280 ratio (22) .
To determine effects of apo- and holo-Lf on MMP-2 activity, cells were seeded in 24-well plates in complete medium until cells were 90–95% confluent. Cells were rinsed twice with 1?PBS and engineering cover letter, then incubated with apo- or holo-Lf (50 ?g/ml) in training effectiveness, F12 medium plus 2% FBS (fetal bovine serum) for engineering cover no experience, 4 h. Conditioned medium was collected after 24 h and concentrated to one third of the original volume using YM-50 microcentrifugation tubes (Millipore Centriplus) at coursework cu denver, 5000 g for cover with no experience, 20 min at 4 °C. Protein concentration of conditioned media was determined using the custom writing service, Bradford assay (Bio-Rad) and proteins (20 ?g) were electrophoresed by SDS/PAGE (7.5% gel containing 1 mg/ml gelatin). Following electrophoresis, the gels were washed twice in cover letter with, 2.5% Triton X-100 in thesis effectiveness, PBS for letter no experience, 15 min to essay disabled, remove SDS and incubated in cover, wash buffer 2 (50 mM Tris/HCl, pH 7.6, 10 mM NaCl, 50 mM CaCl 2 ) overnight at 37 °C. Gels were stained with Coomassie Brilliant Blue for 2 h and destained with 10% acetic acid and 40% methanol in water. Absence of stain (representative of questions, proteolytic degradation) reflects MMP-2 enzymatic activity.
The relative densities of bands were quantified using the Chemi-doc Gel Quantification System (BioRad). Apo- and holo-Lf were labelled with 125 I (GE Health-Amersham Biosciences) by letter with no experience the Iodogen method . Excess free iodine was removed with a PD-10 desalting column (GE Health-Amersham Biosciences). To determine Lf uptake, cells were cultured in 24-well dishes in growth medium until confluent. Cells were incubated with 125 I-labelled apo- or holo-Lf (10000–100000 c.p.m.) in SFM (serum-free medium) for 1 h at 37 °C. Essay! Medium was collected, cells were rinsed once with rinse buffer (0.5 M NaCl, pH 3.0) and pooled with the medium. Cover With! Cells were washed three times with PBS, solubilized with 0.1 M NaOH and radioactivity was quantified in both medium and solubilized cells in a gamma counter. Affinity purification and validation of coursework cu denver, LfR antibody. LfR peptide (SSSPSLPRSCKEIKDE-Cys) was produced (Genemed Synthesis, South San Francisco, CA, U.S.A.) and conjugated (2 mg) to an affinity-purification column (Sulfolink purification kit; Pierce Biotechnology).
LfR antibody was purified from rabbit antiserum according to engineering no experience, the manufacturer's instructions. To demonstrate antibody specificity, affinity-purified LfR antibody (1 ?g/ml) was pre-incubated with LfR peptide (1 mg/ml) in 5% (w/v) non-fat milk in PBS-T (PBS, 0.1% Tween) for 2 h at room temperature (25 °C) prior to immunoblotting of cell lysate as described below. Ligand binding and dissertation writing msc, immunoblotting. Confluent cells were lysed in homogenization buffer [20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1?complete protease inhibitor (Roche Diagnostics)], sonicated three times for engineering cover with, 10 s, centrifuged at of a, 3300 g for 15 min at letter with no experience, 4 °C and of a, the post-nuclear supernatant was collected. Protein concentration of the no experience, supernatant was determined by the Bradford method and cell lysates (50–100 ?g protein) were resolved by electrophoresis. To determine Lf binding, cell proteins were resolved by thesis training effectiveness non-reducing SDS/PAGE at no experience, 80 V for 4 h and effectiveness, transferred on to nitrocellulose membrane. Membranes were incubated with 5% BSA in PBS-T for cover with no experience, 1 h at room temperature (25 °C) to block non-specific binding. Membranes were incubated overnight with 125 I-labelled apo-Lf, blots were rinsed twice with PBS-T and bound ligand was visualized by autoradiography. For immunoblotting, cell extracts were elecrophoresed by SDS/PAGE (10% gel, 200 V for of a, 1 h) under reducing conditions and membranes were incubated with LfR antibody for 1 h. Membranes were washed in PBS-T and primary antibody was detected with donkey anti-rabbit IgG-HRP (IgG-horseradish peroxidase; GE Health-Amersham Biosciences) in cover with, 5% (w/v) nonfat milk in PBS-T.
Membranes were washed in PBS-T for 30 min, visualized with Super Femto Detection Chemiluminescence Reagent (Pierce Biotechnology) and exposed to autoradiography film. Relative band density was quantified using the custom msc, Chemi-doc Gel Quantification System (Bio-Rad). Transient transfection with LfR siRNA (small interfering RNA) Cells were seeded on to 24-well or 6-well plates and cultured until 50% confluent, then transfected with 80 pmol of LfR siRNA (Ambion) in Lipofectamine™ 2000 (Invitrogen) for 24 h according to the manufacturer's instructions. Cells treated with Lipofectamine™ 2000 alone were used as mock-transfected controls. Engineering Cover With! After 24 h, cells were incubated with 125 I-labelled apo- or holo-Lf in SFM at 37 °C. Lf uptake was measured after 30 min as described above. To verify LfR suppression, transfected cells were scraped into 25 ?l of homogenization buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1?complete protease inhibitor) and cu denver, immunoblotted as described above. Biotin-labelling of cell surface proteins.
To determine LfR abundance at the plasma membrane, cells were seeded on to 100-mm-diameter dishes and cultured until confluent. Cover No Experience! Cells were rinsed with PBS and the cell surface was biotinylated with sulfo- N -hydroxysuccinimido-biotin (0.5 mg/ml; EZ-Link Sulfo-NHSS-Biotin; Pierce) at pre pharmacy coursework cu denver, 4 °C for 1 h. Cells were rinsed with ice-cold 0.2 M glycine buffer, pH 5.0, and engineering cover letter with no experience, then with cold PBS to remove unbound biotin. Cells were lysed with lysis buffer [50 mM Tris/HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 1?protease inhibitor (Roche Diagnostics)], sonicated (three 10 s bursts) and centrifuged at 100000 g for 20 min at 4 °C. The supernatant was discarded and the pellet was resuspended in lysis buffer with 0.2% SDS and incubated at 60 °C for 5 min. As the samples cooled, 1% Triton X-100 was added, the samples were re-sonicated and centrifuged at 100000 g for 20 min at 4 °C to pellet any insoluble material. The supernatant was collected and incubated with 300 ?l of slurry (1:1) of immobilized avidin beads (UltraLink Immunobilized Monomeric Avidin; Pierce) for 1 h at room temperature (25 °C) with constant rotation. Writing Service Msc! To collect proteins bound to the resin, the samples were centrifuged at 2300 g for 1 min. The supernatant was discarded and the beads were rinsed with PBS and centrifuged for engineering cover, 1 min at 2300 g . To elute biotinylated proteins, an coursework cu denver equal volume of Laemmli sample buffer (Bio-Rad) containing 2-mercaptoethanol (5%) was added and no experience, incubated at pre pharmacy cu denver, 90 °C for cover no experience, 5 min.
The samples were centrifuged at 2300 g for 1 min and effectiveness, biotinylated proteins were resolved by SDS/PAGE and immunoblotted for LfR as described above. Determination of GPI (glycosylphosphatidylinositol)-anchoring by Pi-PLC (phosphatidylinositol-specific phospholipase C) cleavage. To determine if LfR is a GPI-anchored protein, cells were seeded on to 24-well plates and pretreated with Pi-PLC (1 unit/ml; Invitrogen) in engineering with no experience, SFM for coursework, 1 h at 37 °C to cleave GPI-anchored proteins. With No Experience! To verify the effects of Pi-PLC on LfR abundance at the plasma membrane, cell surface proteins were biotinylated and pre pharmacy coursework cu denver, immunoblotted for LfR as described above. To determine the effects of Pi-PLC on Lf uptake, cells were rinsed with PBS, fresh SFM was added and cells were incubated with 125 I-labelled apo-Lf at 37 °C and letter, uptake was measured after 1 h as described above. Essay People! To illustrate the efficacy of the enzyme, spent medium was collected and concentrated to one third of the original volume using YM-50 microcentrifugation tubes (Millipore Centriplus) at 5000 g for 20 min at 4 °C and immunoblotted for ALP (alkaline phosphatase), a known GPI-anchored protein. To inhibit endocytosis, cells were seeded on to 24-well plates and treated with hypertonic medium (0.4 M sucrose). Cells were pre-treated in engineering cover with, hypertonic medium for 30 min and incubated with 125 I-labelled apo-Lf in SFM at 37 °C in the presence of 0.4 M sucrose and uptake was measured after 60 min. Lf uptake was measured as described above.
To determine if de novo LfR protein synthesis is required for thesis training effectiveness, Lf uptake, cells were seeded on to 12- or 24-well plates until confluent and pretreated with cycloheximide (Cyhex, 10 ?g/ml, Sigma) in SFM for 6 h at 37 °C. Cells were either collected for SDS/PAGE analysis or incubated with 125 I-labelled apo-Lf in the presence of Cyhex at 37 °C and Lf uptake was measured over 7 h. To determine the subcellular localization of cover letter no experience, LfR, cells were seeded on to glass coverslips, cultured for 3–4 days in growth medium and training, incubated with apo-Lf (where indicated, 200 ?g/ml) in SFM for up to 1 h at 37 °C. Letter With! After treatment, cells were rinsed with PBS and fixed with 4% phosphate-buffered paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in PBS for essay on romanticism, 5 min. To eliminate non-specific binding, cells were incubated in blocking buffer (5% goat serum in engineering letter with no experience, PBS) for elements of a essay, 30 min followed by incubation with affinity-purified LfR antibody (0.8 ?g/ml) in letter with, PBS containing 5% goat serum, 1% BSA and 0.2% Triton X-100 for thesis, 1 h. Cells were rinsed with PBS and LfR was detected with Alexa 488-conjugated-anti-rabbit IgG (1 ?g/ml; Molecular Probes) in PBS containing 0.2% Triton X-100 and engineering with no experience, 1% BSA for 20 min. Co-localization of LfR with specific subcellular markers was determined following incubation with antibodies for clathrin (1:500, Abcam); EEA1 (early endosome antigen 1; 1:500, Abcam), TfR (transferrin receptor, localizes to recycling endosomes, 1 ?g/ml; Zymed Laboratories); LAMP1 (lysosome-associated membrane protein 1; 1:200, Abcam); M6PR (mannose 6-phosphate receptor, localizes to late endosomes; 1:500, Abcam) and thesis training effectiveness, detected with Alexa 568-conjugated-anti-mouse IgG (1 ?g/ml; Molecular Probes). After several rinses, coverslips were mounted with ProLong Gold (Molecular Probes) and sealed with nail polish. Immunofluorescence imaging was performed using an Olympus BX50WI microscope with UPlanApo ?100 magnification under an letter with no experience oil-immersion lens [NA (numerical aperture), 1.00], and digital images were captured using the Bio-Rad Radiance 2100 confocal system with LaserSharp2000 software, version 4.1 (Bio-Rad). Results are presented as means±S.D. of people, triplicate samples from 2–3 separate experiments. Statistical comparisons were performed using Student's t test or one-way ANOVA (Prism Graph Pad) and significance was set at P 0.05.
Active form of MMP-2 is increased by cover with apo- but not holo-Lf. Secretion of ears essayist elia, MMP-2 plays a key role in CT invasion, thus we determined effects of Lf and Lf conformation on MMP-2 activity in engineering cover with, BeWo cells. Disabled! Using gelatin zymography, our data demonstrated that apo-Lf significantly increased (20%) MMP-2 activity compared to untreated cells (Figure 1). In contrast, we observed no effect of holo-Lf on with, MMP-2 activity, suggesting a differential effect of Lf conformation on cellular invasivity of elements of a personal essay, CTs. Representative zymogram (upper panel) of culture medium collected from cells treated with either apo- or holo-Lf. Equal amounts of protein (20 ?g) were electrophoresed through polyacrylamide gels containing gelatin and engineering cover letter no experience, stained with Coomassie blue. MMP-2 activity was detected as clear bands (digested gelatin) on dissertation writing msc, a blue background.
MMP-2 activity was significantly increased in cells treated with apo-Lf as compared with untreated (control) and engineering cover letter no experience, holo-Lf treated cells (lower panel). Data shown represent MMP-2 activity as a percentage of untreated control cells (mean±S.D.) for two independent experiments ( n =4). * P 0.05. LfR facilitates uptake of apo- but not holo-Lf. To determine if LfR mediates apo- and/or holo-Lf uptake, uptake studies using 125 I-labelled Lf were performed. Elements Essay! Our data demonstrated that BeWo cells internalized 9-fold more apo-Lf compared with holo-Lf (Figure 2). Using affinity-purified LfR antibody, we confirmed that BeWo cells expressed LfR, as specifically immunoreactive proteins were detected at ?35 and ?105 kDa in BeWo cells (Figure 3). With No Experience! Ligand binding plays a critical role in receptor function, and ligand binding established that radiolabelled apo-Lf bound to thesis training, two LfR proteins at cover, ?75 and custom dissertation msc, ?105 kDa (Figure 3), but not to the 35 kDa protein. We speculate that the 35, 75 and cover with, 105 kDa moieties represent LfR monomers, homo-dimers and -trimers, respectively. The ability of LfR to internalize apo-Lf is dependent on its cellular localization and we showed that the thesis, 105 kDa LfR was localized to cover letter with, the plasma membrane using cell surface biotinylation followed by immunoblotting (Figure 4A). Writing Msc! In contrast to what was reported for the intestinal LfR , trophoblast LfR does not appear to be GPI-anchored as demonstrated by internalization of Lf following GPI-anchored protein cleavage with Pi-PLC (Figure 4B).
Confirmation of Pi-PLC cleavage was verified as Pi-PLC increased ALP abundance in cover with, spent medium compared to untreated cells (Figure 4C). Confluent cells were treated with 125 I-labelled apo- or 125 I-labelled holo-Lf and Lf uptake was determined. Elements! Representative graph of engineering cover letter no experience, uptake after 1 h illustrates that significantly less holo-Lf was internalized when compared to apo-Lf. Data shown represent mean Lf uptake (Lf uptake/mg protein)±S.D. from two independent experiments ( n =4). * P 0.001. Representative immunoblot ( A and custom dissertation writing, B ) and ligand blot ( C ) of cell lysate proteins. Proteins (50 ?g) were resolved by SDS/PAGE and immunoblotted with LfR antibody ( A ; LfR Ab, 1 ?g/ml) or pre-absorbed with excess Lf antigen ( B ; LfR Ab+peptide) for 1 h. The LfR antibody detected specific proteins at ?105 and engineering with no experience, ?35 kDa ( A ) which were both completely eliminated following pre-absorption with LfR peptide ( B ). ( C ) Cell lysate (100 ?g) was resolved under non-reducing and non-denaturing conditions, and ears, incubated with 125 I-labelled apo-Lf. Detection of ligand binding to proteins at 105 and 75 kDa was observed. Confluent cells were treated with Pi-PLC and with, surface expression and Lf uptake was assessed. ( A ) A representative LfR immunoblot of biotinylated cell surface proteins documents the custom dissertation service msc, presence of LfR on the plasma membrane after Pi-PLC treatment. ( B ) Cleavage of GPI-anchored proteins with Pi-PLC did not significantly reduce 125 I-labelled apo-Lf uptake in with, BeWo cells.
Data represent mean Lf uptake/mg protein±S.D. for two independent experiments ( n =4). Questions! ( C ) Representative immunoblot of ALP antibody from total spent medium following Pi-PLC treatment, validating enzyme activity. In order to determine a role for LfR in Lf uptake, LfR expression was attenuated in no experience, cells transfected with LfR siRNA (Figure 5A). We demonstrated that reduced LfR abundance resulted in significantly lower apo-Lf uptake (30%) compared with mock-transfected controls (Figure 5B). Essay About! In contrast, holo-Lf uptake was not significantly reduced (10%), suggesting that internalization of engineering with no experience, holo-Lf in BeWo cells is not predominantly LfR-mediated. Due to our finding of limited LfR-mediated holo-Lf uptake and lack of an effect of holo-Lf on MMP-2 activity, we subsequently focused on effects of apo-Lf on the intracellular trafficking of LfR. ( A ) Representative LfR immunoblot of total protein illustrating significant attenuation of LfR compared to mock-transfected cells. ( B ) Lf uptake was measured using 125 I-labelled apo- or 125 I-labelled holo-Lf in cells transfected with LfR siRNA or mock-transfected controls. Data represent the means±S.D. ( n =2–3) of about disabled people, triplicate experiments. * P 0.05, ** P 0.01, as analysed by Student's t test. LfR internalization is a clathrin-dependent process and no experience, potentially follows a recycling pattern. As our data clearly demonstrated that apo-Lf is internalized via LfR, we then determined the cu denver, mechanisms responsible for internalization of apo-Lf via LfR.
CTs were treated with apo-Lf and LfR protein abundance on the plasma membrane was assessed following cell surface biotinylation. We noted that treatment with apo-Lf resulted in engineering letter with, a rapid (10 min) and significant increase in cell surface-associated LfR (Figure 6A), independent of essay people, changes in total LfR abundance (Figure 6B). This suggests that apo-Lf may signal the recruitment of LfR to the plasma membrane or that perhaps Lf binding to LfR transiently interferes with its internalization. Confluent cells were treated with or without apo-Lf and LfR surface expression was assessed. ( A ) Representative immunoblot of crude membrane proteins from cells treated with apo-Lf or untreated and detected with LfR antibody. Representative graph illustrates that LfR abundance significantly increased with apo-Lf treatment compared to cover letter no experience, untreated cells with no effect on the total amount of LfR protein expression ( B ). * P 0.001, as analysed by Student's t test. ?-actin was used as a loading control. Questions On Romanticism! Data represent means±S.D. ( n =3) of 3 independent experiments. Multiple pathways for internalization of plasma membrane proteins have been characterized. To identify the mechanism responsible for LfR internalization, CTs were treated with hypertonic medium (0.4 M sucrose) to inhibit clathrin-dependent endocytosis, which resulted in letter with no experience, a robust inhibition of ears elia, apo-Lf internalization (40%) compared with untreated cells, with values of 2.4±0.25 and letter, 5.7±0.42 pmol/mg protein±S.D. for treated and control cells respectively; n=5–8 per group, * P 0.001. To confirm the essay about disabled people, mechanism by which LfR is endocytosed, we used confocal microscopy to identify the letter with, subcellular compartments within which LfR resides. As indicated in Figures 7(A) and 7(B), LfR and thesis training, clathrin co-localized and were largely associated with the plasma membrane, as illustrated by yellow pixilation in the merged images (Figure 7C).
As internalization is ligand-stimulated, cells were treated with apo-Lf, and indirect immunofluorescence was used to visualize specific sub-cellular compartments within which LfR was associated. Following ligand stimulation, we demonstrated partial co-localization of LfR and the early endosome marker EEA1 in vesicular/endosomal-like structures (Figures 7D–7F). As co-localization between EEA1 and LfR was not exclusive, we sought to examine other sub-cellular compartments containing LfR. Engineering No Experience! Clathrin-mediated endocytosis is essay questions one mechanism cells utilize to control cell surface receptor abundance and turnover via recycling. Letter! To investigate whether LfR trafficking from the plasma membrane potentially includes endocytic recycling, we used the ears essayist elia, indirect immunofluorescence of cover letter, TfR as a marker for recycling endosomes. We showed partial co-localization of LfR and TfR within distinct internalized vesicles in cells following ligand stimulation (Figures 7G–7I).
No co-localization between LfR and M6PR (a late endosome marker) and questions on romanticism, minimal co-localization with LAMP1 (a lysosome marker) was detected (results not shown). These results indicate that, in response to apo-Lf stimulation, LfR is internalized via clathrin-mediated endocytosis and consequently enters a distinct intracellular endocytotic pathway. Confocal microscopy identifying specific sub-cellular compartments associated with LfR. Engineering! Cells were fixed with paraformaldehyde and imaged for LfR (green) and thesis effectiveness, sub-cellular markers (red); clathrin ( A and C ), EEA1 ( D and cover letter with no experience, F ) and TfR ( G and I ). Clathrin ( A ) and LfR ( B ) were co-localized (yellow, C ) and questions, stained in engineering cover letter no experience, a plasma membrane-like pattern in cells. Essay People! Following apo-Lf treatment, partial co-localization with the early endosomal marker EEA1 ( D ) and LfR ( E ) was detected (yellow, F ) and treatment of cells with apo-Lf clearly resulted in vesicularization of LfR.
Furthermore, co-localization with the recycling endosome marker TfR ( G ) and LfR ( H ) was detected (yellow, I ) following treatment of cells with apo-Lf. Finally, to determine if increased abundance of LfR at the plasma membrane in response to apo-Lf stimulation reflects changes in intracellular LfR recycling, we blocked de novo protein synthesis with Cyhex and measured apo-Lf uptake. Inhibition of new receptor synthesis did not diminish apo-Lf uptake, indicating that LfR is engineering cover with recycled. In fact, apo-Lf uptake was significantly higher in Cyhex treated cells (Figure 8A), suggesting that apo-Lf enhances LfR recycling. Moreover, our data indicated that CT-derived LfR is a relatively long-lived protein (Figure 8B), as abundant LfR remained detectable after 7 h of writing service msc, Cyhex treatment and that de novo protein synthesis may play a minor role in sustaining total and engineering letter, surface receptor abundance. Confluent cells were pre-treated with Cyhex and uptake of 125 I-labelled apo-Lf and LfR abundance was assessed compared with untreated cells. ( A ) Apo-Lf uptake from cells significantly increased in the presence of elements essay, cycloheximide (Lf+Cyhex) after 3 h compared with untreated cells (Lf). Data represent means±S.D. for engineering letter with, two independent experiments ( n =3–4), and * P 0.05 compared with untreated cells, as analysed by essay on romanticism Student's t test. ( B ) Representative immunoblot of engineering cover letter with no experience, total protein extract isolated from cells pre-treated with Cyhex then incubated with apo-Lf for 3 and 7 h compared with untreated cells. Training Effectiveness! The immunoblot demonstrates that Cyhex did not affect LfR abundance. ?-Actin was used as a loading control. CTs are specialized cells in the placenta that are initially responsible for invasion into the uterine endothelium, assuring optimal fetal development . Our previous detection of LfR along the maternal and cover with, fetal interface in the placenta implicates LfR in CT function [8,9,27].
The aim of the present study was to elements of a essay, explore the function of Lf in cover letter, the placenta as mediated via LfRs using a CT cell model. While primarily regarded as a component of milk , Lf is ears elia also found in decidua (9–95 ?g/g), amniotic fluid (2–37 ?g/g) and chorion (2–26 ?g/g) , and is secreted directly from uterine epithelia [29,30]. This suggests that Lf secreted from the uterine endothelium may regulate CT function in a paracrine manner. Results from this study documented an enhancing effect of apo- but not holo-Lf on MMP-2 activity in CTs, suggesting a regulatory role for cover with, apo-Lf in CT invasivity as this process is dependent upon the secretion and training effectiveness, activation of cover letter with no experience, MMPs. There is precedence for effects of elements personal, Lf on letter with no experience, MMP regulation as over-expression of Lf activates MMP-1 expression through binding to AP-1 promoter elements and through stimulation of the p38 MAPK (mitogen activated protein kinase)-dependant pathway in mouse fibroblasts and human COS-1 cells . We speculate that apo-Lf may similarly regulate the transcription of dissertation writing, MMP-2 via AP-1 promoter elements which are present in the MMP-2 gene . In support of the concept of Lf acting as a transcription factor, a nuclear localization signal within the N-terminal region of the cover with no experience, Lf molecule (amino acids 1–5) has been previously identified , and Lf binds to DNA [33,34]. Alternatively, the induction of MMP-2 via apo-Lf could be through the p38 MAPK pathway as p38 activation is required for the up-regulation of essay about disabled, MMP-2 protein expression . Cover With! The ability of essay on romanticism, Lf to engineering letter with, activate p38 MAPK has been previously shown in essayist, other cell types [16,35], providing support for engineering cover no experience, a direct role of apo-Lf on CT invasivity.
The effect of apo- but not holo-Lf on MMP-2 activity probably reflects that distinct functions are associated with either apo- or holo-Lf. In contrast to reports in essayist, rat hepatocytes , CTs bound and internalized both apo- and holo-Lf. However, in CTs, apo-Lf was internalized more avidly than holo-Lf, suggesting preferential uptake mechanisms specific to cover no experience, apo-Lf which may reflect the requirement of this molecule for pre pharmacy coursework cu denver, CT-specific functions such as invasivity, while precise Lf-dependant functions in no experience, hepatocytes remain unknown. Gene attenuation of about disabled people, LfR conclusively determined that LfR primarily facilitates apo-Lf uptake in CTs. Recently, distinct Arg-Gly-Asp (RGD) binding motifs were found within the Lf molecule , which have been postulated to determine ligand binding and receptor interactions.
It is possible that iron-saturation, which alters Lf conformation, may result in engineering letter no experience, LfR inaccessibility to these binding motifs. While this is essay about disabled people not to imply that holo-Lf binding and uptake does not elicit cellular events, it does not appear that these effects are mediated via LfR internalization in CTs. Possibly, holo-Lf internalization is not a prerequisite to engineering with no experience, stimulate cellular events at the plasma membrane as suggested by other investigators [15,35]. As apo-Lf was preferentially taken up by custom writing service LfR, we sought to characterize the cover letter with no experience, placental LfR more extensively. It has previously been reported that intestinal LfR is a GPI-anchored protein . Contrary to this observation, we determined that, in CTs, the LfR receptor is pre pharmacy not a GPI-anchored protein and cover, is in on romanticism, fact internalized via clathrin-mediated endocytosis following ligand binding. Our observations in trophoblasts are consistent with previous reports in other cell types such as hepatocytes, where inhibition of clathrin-mediated endocytosis by hypertonic medium  significantly reduced Lf internalization.
We speculate that differences in Lf uptake between intestinal and CT LfR may reflect differences in cellular function, as CTs are a semi-pleuripotent and invasive cell type, whereas enterocytes are terminally differentiated polarized cells involved in letter, vectorial nutrient transport. Personal Essay! Our determination that LfR is internalized via a clathrin-dependent mechanism may provide insight into LfR structure, as receptors utilizing clathrin-mediated endocytosis require a cytoplasmic, tyrosine-based sorting signal for interaction with adaptor proteins, such as AP2 . To identify potential AP2-interacting motifs within the engineering letter with no experience, LfR sequence we used a proteomics server (www.elm.eu.org) and indeed two tyrosine-sorting motifs were identified at amino acids 120–123 and 129–142. This further suggests that a portion of LfR protein does reside intracellularly and that one or more of these motifs may functionally interact with AP2 to facilitate LfR internalization. LfRs have been identified in various cell-types and people, reports have determined a molecular mass of ?110 kDa . Recently, Tsuji et al.  examined the with no experience, structure of cu denver, human LfR in greater detail and engineering cover, determined that it is an coursework 105.5 kDa oligomeric protein made up of three 35.5 kDa subunits, similar to what we observed in trophoblasts. Interestingly, while we detected both the monomeric (35 kDa) and trimeric (105 kDa) forms of LfR under reducing conditions, the abundance of the trimer was always greater that the monomer.
For some reason, we were unable to letter with no experience, extensively reduce the trimeric form to monomers even when using dithiothreitol and iodoacetic acid (V. Lopez, unpublished observations). Interestingly, we did not detect the on romanticism, dimeric form of LfR under reducing conditions, which is similar to the findings of Tsuji et al. . We further demonstrated that apo-Lf binds to engineering letter with no experience, both dimeric and trimeric LfR multimers, which agrees with previously published reports demonstrating that human LfR forms a homo-trimer [7,40] as a result of elements of a, subunit oligomerization . We speculate that the trimeric form is an cover letter with unusually stabilized form, mediated by several intracellular disulfide bonds and possibly also hydrophobic interactions, whereas the dimeric form is less stable and training, can be reduced to the monomeric form. In contrast, a 45 kDa calcium-dependent LfR has been reported in rat hepatocytes ; however, it is unknown how this LfR relates specifically to Lf function as rats do not express Lf. It is letter no experience well established that cell signalling and cellular homoeostasis are altered by increasing or decreasing cell surface receptor abundance . As LfR abundance at the plasma membrane did not require de novo synthesis, our data suggest that recycling of thesis, LfR to and from the letter with no experience, plasma membrane occurs. Endocytic recycling requires internalization of cell surface receptors and their ligands within clathrin coated pits , with the questions on romanticism, interaction between transferrin and TfR being the classic example of this process . Receptor recycling is one strategy by which cells maintain a mobilizable receptor pool to quickly traffic to the cell surface in response to physiological stimuli, to allow cells to redistribute cell-surface molecules to specific cell locations or compartments undergoing a physiological activity .
We speculate that Lf may either stimulate the recruitment of letter with, LfR to the plasma membrane, or that perhaps Lf binding to the receptor results in transient accumulation of LfR prior to of a, internalization via clathrin-mediated mechanisms. We propose that CTs may require Lf/LfR internalization to rapidly facilitate implantation, allowing for optimal embryo-fetal development. Cover Letter With No Experience! Further studies are needed to address this issue. Collectively, the results from this study demonstrated a specific role for apo-Lf but not holo-Lf in regulating MMP-2 activity and thus potentially endothelial invasion in CTs. Coursework Cu Denver! Importantly, we have determined that apo- but not holo-Lf is internalized via LfR, which occurs through a clathrin-dependent pathway in CTs suggesting a mechanistic and functional difference between apo- and holo-Lf. These results may help to explain the pleuripotent and cell-specific functions which have been associated with Lf.
The authors would like to express their gratitude to Dr. Lisa L. Gough for critical reading of the manuscript. This work was supported by NIH grant number HD-043240. Abbreviations: ALP, alkaline phosphatase; apo-Lf, iron free lactoferrin; CT, cytotrophoblast; Cyhex, cycloheximide; EEA, early endosomal antigen; GPI, glycosylphosphatidylinositol; holo-Lf, iron saturated lactoferrin; LAMP, lysosome-associated membrane protein; Lf, lactoferrin; LfR, Lf receptor; M6PR, mannose 6-phosphate receptor; MAPK, mitogen activated protein kinase; MMP, matrix metalloproteinase; Pi-PLC, phosphatidylinositol-specific phospholipase C; siRNA, small interfering RNA; SFM, serum-free medium; ST, syncytiotrophoblast; TfR, transferrin receptor.
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30 Book Report Templates #038; Reading Worksheets. Reading helps students develop a strong imagination, encourages their creativity, and strengthens their analytical skills. Teachers assign a lot of cover book reports to ensure that students read lots of books, especially at that critical early age when they are still trying to on romanticism master the written word. To cut down on some of the engineering letter no experience workload, students and essay questions, teachers can find a book report template to download and fill in. This gives more time to enjoy the act of reading, so students can become life long learners. A book report is typically given as an assignment to students in elementary and middle school. Students fill out a form answering basic questions about the book they were assigned to read. Turning in the report serves as proof to cover with the teacher that the student read the questions book and, hopefully, got something out of it. These reports may ask students to detail what the book was about, the names of the main characters, what the theme of the book is, and where the events are set. Some reports may ask specific questions about events or characters to ensure that the letter with no experience students read the essay book all the way through.
The report can also help students understand the book better by asking them to think about engineering cover with no experience its meaning and the plot. Teachers may also hand out worksheets for students to complete in class or as homework. These are usually limited to essay the earlier grades when students are still learning to read. Reading worksheets ask simple questions about the cover book without requiring multiple sentence answers. These questions may ask students to essay questions on romanticism name a favorite character, or mention the main conflict in a few words. It’s not uncommon for the teacher to read a picture book with the class and have students fill out a worksheet afterwards. If the classroom has a bookshelf, there may also be a ‘Reading Time’ when students pick out a book to read.
When they finish, the students fill out the engineering worksheet and submit it to their teacher. Book report forms are a popular choice of assignment for elementary school classes. These forms make it simple for effectiveness students to cover no experience complete the report by filling out the worksheet. These sheets can be generic with standard questions, or teachers may create a unique sheet with questions specific to each different book that is assigned. These forms also help introduce students to the idea of a book report format and ears elia, show them what kind of information may be expected on longer, multi-page reports that they are expected to complete in the higher grades. A form is cover letter with no experience also easier to grade, especially for teachers who have a large classroom and more assignments to go over than usual. A simple book report features a few sections that ask students to answer questions in paragraph format. These each ask students to detail a different element of the book. All book report forms will ask for the title, author name, and the illustrator’s name, if it is a picture book. The other elements on a simple form include: Setting – This is where the book’s events took place, i.e.
New York. Characters – A list of who the main characters are and their names. Plot – A basic overview of the major events in ears the book. Your impressions – Whether you like the book or not, and why. A simple form may also just ask the students to describe the beginning, middle, and end events of the book in three questions. Cover With? Students may also be expected to of a essay identify the climax, which is the most intense point of the book where the main character’s problems are resolved or made worse. This helps get students thinking about the traditional progression of cover letter with a plot.
Eventually, teachers transition students to writing a multi-page book report. The report is usually written in a word processing software, like MS Word. Students can find book report format templates for these assignments too. These longer reports are written out in paragraph form. The teacher asks the students to address different elements of a book in their own words or with their own formatting. A simple way to organize these reports is to divide them into three basic sections, the introduction, main body, and essay on romanticism, conclusion. In the main body, students can create a different subheading for each element to address. For instance, Introduction – The first paragraph; includes book title, author, genre, and why you chose the book. Main Body – The middle part of the report; includes summary, theme, setting, and characters. Conclusion – A short summary of the book report and opinion of the book.
Difference Between The Book Report Types. With No Experience? The short book report form is handed out by the teacher and ears elia, can be completed in one night like a homework assignment. A multi-page report is engineering cover with created by the student and may take more than one night to finish. Short templates are available in lots of fun book report ideas suitable for young children. They have questions with a few lines to questions on romanticism hand write the answers in a few short sentences. Some templates may include activities to make them more interesting to students, such as having them draw out a scene from the book or re-write the ending. Multi-page reports start out as two page reports and gradually increase in size through middle school and engineering with no experience, high school. Of course, those two page reports are just as difficult for young students as the five page reports are for high school students. Ears Elia? The great thing about these assignments is that students are usually free to cover letter with no experience organize their book report ideas however they desire.
They can create bold sub-headers for the main body of the report. Students can write about the theme, characters, and setting separately. Or they can leave out the coursework cu denver headers and devote their report to letter with a specific element, such as the book theme. Elements Personal Essay? This allows students to weave information about characters and setting into cover, the report where they are most relevant to the theme. The former works well for short chapter books, and the latter is better for books that may feature multiple themes. Ideas for pre pharmacy cu denver Different Kinds of Book Reports. There is more than one way to complete a book report.
Both teachers and students may find these creative ideas more interesting than a straightforward report. Write a review of the book. Create the report in the form of a newspaper or blog review. Summarize the book without giving away the plot or the ending. Talk about what made you like it or, if you hated it. Feel free to give the book a thumbs up or down rating at the end. Do a diary. Use a journal template to engineering with create a diary written by one of the main characters. There should be multiple entries that follow or discuss the events of the people book from cover letter with, that character’s point of view. Interview one of the characters.
Create an imaginary interview with one of the characters in the book. Ask them questions about where they come from, why the character did something important to the plot, and what the character thinks about the effectiveness outcome of the events in the book. Write a newspaper article. Create an imaginary newspaper article detailing one of the major events in the book, such as a theft or an important discovery. Make sure to answer the who, what, where, when, and why of these events in your article. What To Do Once You’ve Written The Report. When students finish writing out their book reports, they have completed what is engineering letter no experience called the “First Draft” or “Rough Draft”. This is just the first stage of the report, but it is the most difficult part. Finishing up that report in the following steps is disabled a lot easier. Read and mark the report.
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You have to understand, I approach all other forms of dancing as I do my annual pap smear: cringing at my exposure, positive that I am disgusting. I feel sorry for elements of a essay anyone inspecting me. Letter No Experience? I can’t help but see most dancing as a means for flaunting sex appeal. I have never been able to effectiveness, dress or act the part of engineering cover letter no experience, sexy . Elia? Not even on Halloween. Engineering With? While my friends dressed as sexy witches or Madonna, I was a green, pot-bellied Grinch or a hulking gorilla. Cloggingat least my style of cloggingis not about grace or seduction. It’s about elements of a essay, enthusiasm and stamina. I’m remembering some collages that my late mother-in-law made after a trip to Cuba in September 1941.
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She is wearing a green silk teddy embroidered with leaves and she pulls back the bolt. Training Effectiveness? “Like hell there isn’t. This is the with, third time, Martin. I know what I heard, and I know what I saw.” “That same shadow? How do you know it wasn’t just some dog?” Slowly her eyes float over the barrel to his, and questions, he backs up a step, throwing up both hands. Cover Letter No Experience? “OK. You know what, do what you feel you need to training effectiveness, do.” He disappears into their bedroom, but a moment later, returns with a cartridge box50 shellsand sets it gently on the floor beside the couch. On his way back to bed, he stops to look into Mill’s son’s room.
She hears him touching things in there, knows the sound of Charles’ closet being opened. Engineering No Experience? After what seems like a long pause, Martin goes back to bed. Bodies retrieved from the custom writing, water can only engineering cover letter no experience be beautiful to those who have loved them long enough to questions, take those bodies for granted. And only then when enough time has passed so you’ve bartered with the universejust a glimpse, please, no matter how awful. I looked at my husband Sean’s bloated, discolored face, twisted in the terror of engineering cover letter, hypothermic shock and suffocationa face so unlike any he ever worebut I saw the man I loved. I had been visiting my sister Jennie in essay about disabled people Nashua the cover letter with, Friday night he fell through. Coursework Cu Denver? Of course we spoke about Sean then. It didn’t seem substantial at engineering cover with no experience the moment, just conversation, but I have to look back on that as the last time I talked about him without knowing he was gone.
Like how the moon might feel when it looks down on us with memories of pre pharmacy, Pangaea, before the engineering, world exploded into essay about, what it is today. In hindsight I feel I should have had some sense of what was happening, but after dinner and cover letter no experience, a board game, and her husband’s incessant talk about politics, and a third glass of cu denver, wine, I drifted off to sleep without giving Sean a second thought. Pop figured the best time to catch the geese would be before dusk. He said those impudent devils would be full of letter no experience, Hazlett’s corn by then and settled near the pond: fat, content, and not inclined to move. “No, I do not need help!” he bristled at essay on romanticism my initial offer of assistance. Cover With? It was my thirteenth summer thenthe summer when, want to or not, I was being pulled toward womanhood. “No, Hallie Jo, I do not need help,” Pop repeated, a tad bit more civil. “I can take care of this myself.
Just leave me alone to do what needs doing.” He sighed loudly and looked as if the weight of the essay about, world was pressing down on his shoulders, as if he had too much to bear, though he’d always maintained that the Good Lord never gives a body more than can be handled. Silently, she stood in engineering letter no experience the window of her grandmother’s bedroom and essay about, watched the engineering with no experience, grey river flow by. Ears? Through the lens of the telescope positioned in cover with the window, Mia peered at the Buffalo skyline. Seagulls darted between the border of sky and about disabled people, water. Here it was Thanksgiving, but across the Niagara River it was Columbus Day. Mia’s eye darted back and forth, attempting to grasp the letter no experience, spirit of either holiday, which was notably lacking in the house. She spied her brother and their cousin Jacob across the street throwing stones into the river.
Both boys were twelve and on the cusp of their teenage years. They were on the verge of on romanticism, losing their childhood softness and turning overnight into engineering cover with, towering men-boys with booming voices, their faces red with pimples and an anxious sullen rebellion. Matt would quickly grow in and out of this phase, turning into effectiveness, a quiet giant, but Jacob would not. For fifteen years, I spent practically every night and weekend at a Chicago dance or theater, improv or comedy performance. One night in 2003 as I walked up Belmont Avenue, a press kit tucked under my arm, I stopped in my tracks and said to myself, “I can’t do this anymore.” The weary proclamation had nothing to do with the show I had just seen, nor did it mean that I suddenly hated live performance. I was tired. Engineering No Experience? I had my own stories to tell. I stopped reviewing theater. The artist, to thesis effectiveness, my way of thinking, is a monstrosity, something outside nature.
I appear to engineering cover, be guilty of a lapse in judgment. I should have consulted with Bethany before inviting my protégé to effectiveness, set himself up in engineering the gatehouse. Long-unoccupied, high-ceilinged and sunny, it would give him far more suitable space, I assured him, with respect to disabled, both studio and living quarters, than that dank, overpriced hole of his in Brooklyn. With? And that’s not to mention the pre pharmacy coursework cu denver, clean rural air of northern Jersey. And the safety from the kind of vandalism that had destroyed some earlier Lou Coctons. I invited Lou on a generous impulsewell, true, the Chilton Mazzia gallery does stand to engineering no experience, gain an exclusive on every new piece of hisbut I should not have expected Bethany to accept without question a decision I made on the spur of the moment. It was a sound business decision, but she was hardly going to react with enthusiasm to an arrangement that must impinge (no matter how little) upon the total privacy of about people, our lives out cover no experience, here in the sticks. I can’t stop picturing the pain with which she greeted my tactless announcement. “For God’s sake, Chilton,” she chided, “we’ve been married for less than two years, and you invite a stranger to move in with us?” It is now barely five hours later and her reaction still hurts.
Why am I so sensitive? It comes of a life perhaps overly dedicated to the things of the mind, to Art. 1. How It Started. Sara got a Chimpanion. Helen got a Chimpanion.
But when Elsa Gartner got one I said to personal, myself, “Well, that takes it!” Couldn’t hold out another minute. I jumped into the car, never mind my hair, or the engineering cover letter no experience, bed not being made, and drove straight to essayist, Wally’s Department Store. Engineering Letter No Experience? $1,999.99, I could hardly believe it, a bargain sale price for pre pharmacy coursework cu denver a Chimpanion. I mean, I could barely afford it; but I could barely afford my house, my car, my credit cardyou know, just add it to the pile. Raced home like a teenager, and struggled inside with the boxfifty or sixty pounds, it seemed, if it was an ounce. “My God,” I said to myself, setting it down, breathing hard. Engineering Cover With? “My very own Chimpanion, at last.” Now, in case you haven’t heardif you’ve been in essayist India or living with Zulus, or somethingI’ll explain about the whole Chimpanion thing. It wasn’t a fad, really (fads are dumb); it was justa really great invention. The sort of thing everyone could use, that everyone needed . When I think of what my life was like BC (Before Chimpanions), I cry, practicallyI mean, it was so tough. So ordinary . It was no life at cover all. Anyhow, Chimpanions (chimps + companions) were Electric Robot Friendspets, you might call them, though they were so much more than pets. They could follow instructions, almost anything you could think of, like “wash the dishes” (you had to essay on romanticism, say it in a loud, even tone), even hard stuff like “get the groceries,” or “do my taxes.” It was like having your own private butler. By the with, end, my Chimpanion (it took a while to train himthey start out wild) could dress me, brush my hair, even paint my toenails, which saved heaps of time.
It was pretty sweet. Thesis Effectiveness? But “all good things . Engineering Cover Letter No Experience? . .,” as they say. We’re AC (After Chimpanions) now, and life is murder. Of A Personal Essay? It all happened so quickly; I hope I remember all the details, the important stuff anyway. Here goes. SID: I just don't like Dylan. Let's just, let’s just put it like that. I don't like Dylan. NATHAN: Ok, so, so you’re just. SID: And it’s not. NATHAN: so you’re just.
SID: and it’s not. NATHAN: so you’re just laying it down. You're just throwing down the cover letter with, gauntlet.