Androgens bind to the androgen receptor (AR) in prostate cells and are essential survival factors for healthy prostate epithelium. Most untreated prostate cancers retain some dependence upon the AR and respond, at least transiently, to androgen ablation therapy. However, the relationship between endogenous androgen levels and cancer etiology is unclear. High levels of androgens have traditionally been viewed as driving abnormal proliferation leading to cancer, but it has also been suggested that low levels of androgen could induce selective pressure for abnormal cells. We formulate a mathematical model of androgen regulated prostate growth to study the effects of abnormal androgen levels on selection for pre-malignant phenotypes in early prostate cancer development.α-reductase activity, select more strongly for elevated AR expression than do normal environments. High androgen actually slightly reduces selective pressure for AR upregulation. Moreover, our results suggest that an aberrant androgen environment may delay progression to a malignant phenotype, but result in a more dangerous cancer should one arise.We find that cell turnover rate increases with decreasing androgen levels, which may increase the rate of mutation and malignant evolution. We model the evolution of a heterogeneous prostate cell population using a continuous state-transition model. Using this model we study selection for AR expression under different androgen levels and find that low androgen environments, caused either by low serum testosterone or by reduced 5α-reductase inhibitors such as finasteride may increase the incidence of therapy resistant cancers.The model represents a useful initial framework for understanding the role of androgens in prostate cancer etiology, and it suggests that low androgen levels can increase selection for phenotypes resistant to hormonal therapy that may also be more aggressive. Moreover, clinical treatment with 5This article was reviewed by Ariosto S. Silva (nominated by Marek Kimmel) and Marek Kimmel. By the time Theodosius Dobzhansky famously claimed that "nothing in biology makes sense except in the light of evolution" , cancer Until recently it was an open question whether insights generated by an evolutionary perspective could be translated to the oncology clinic. However, in the last 10 years a wide variety of potential applications has arisen. Evolutionary models of neoplasia led Gatenby, Gawlinsky and colleagues -14 to hyDespite these contributions, application of an evolutionary perspective to clinical oncology still suffers from a lack of maturity. In particular, the theory of tumor ecology--the theater in which evolution occurs--is critical, yet not well characterized for most tumor types -10. One This particular neoplasm is interesting for a number of reasons in addition to its unique ecological features. It accounts for 25% of new cancer diagnoses and 10% of cancer deaths in American men . Most meα-dihydrotestosterone (DHT) by the enzyme 5α-reductase in the prostate TS), we nWe note that there are a number of published values for testosterone and DHT serum and prostate concentrations for both humans and rats. In , serum TKM are available directly from empirical biological data; these values with references are given in Table λ, U, α, βD, have been estimated from a combination of empirical data and steady state analysis.Values for most of the basic kinetic parameters ka = .14 nM-1 hr -1, kd = .069 hr-1, and KD = .49 nM for testosterone, and ka = .053 nM-1 hr-1, kd = .018 hr-1, and KD = .34 nM for DHT. In Dunning R-3327 rat prostate carcinoma, the KD of 0.5 nM for DHT was similar to normal tissues, but the dissociation half-lives for of 60 hr and 160 hr for testosterone and DHT, respectively, were much longer [Several groups have measured binding kinetics for testosterone and DHT to the AR. Wilson and French  measuredh longer . Neverthh longer .et al. [The AR is unstable in the absence of its ligand. Gregory et al.  studied Rt, which equals R + CT:R + CD:R.In the absence of any direct estimates, we use the following method to estimate the AR concentration within prostate cells. Wilson and French  reportedU, is determined as an empirical function of serum testosterone based upon the data of Wright et al. [α-reductase inhibitor finasteride; this data is shown in Figure α = 0 . We also ignore AR production and degradation and impose the total AR concentration Rt = 16-45 nM, leaving U as the only free parameter. Running the system to steady state then allows U to be determined as a function of TS and Rt, as shown in Figure Rt implies a lower influx U, as when more AR is present, more androgen is bound and thus degrades more slowly. To determine α (5α-reductase concentration) and βD (DHT degradation rate), the data-set of Wright et al. where finasteride was not present is used in conjunction with the empirically determined function U. We have found that setting Rt = 45 nM and using the corresponding function for U gives the best fit to the data. A good algebraic fit to the U curve for Rt = 45 nM isThe influx of free testosterone into the prostate, t et al. , who meaα = 5 mg L-1 and βD = ln(2)/9 hr-1. The half-life of DHT in the rat prostate is reportedly greater than 6 hours [βD = ln(2)/9 hr-1 is consistent with this. Using these parameters, the predicted T and DHT concentration versus the actual concentration as a function of serum testosterone is shown in Figure α-reductase blockade (α = 0) versus actual values for finasteride treatment are also shown in Figure This is also indicated in Figure  6 hours ; a degraet al. [λ, is a constant. This implies that AR production may be positively regulated by free AR, or alternatively and more realistically, negatively regulated by androgen:AR complexes.This parametrization shows that when the total AR concentration is a constant, a good fit to the data can be made, and suggests that AR production is regulated according to some homeostatic set point. We have been unable to favorably compare our results to the data of Wright et al.  if AR pret al. found that subsequent reintroduction of androgen rapidly increases the concentration of nuclear AR bound androgen, and the AR protein level rebounds to the pre-castration level within 2 days [et al. found that cytosolic AR was replenished within 3 hours of testosterone reintroduction, and proposed that testosterone regulates AR levels through its effect upon receptor half-life [Indeed, AR mRNA and protein levels are clearly regulated by androgens, and this has mainly been studied in rat models of castration and androgen reintroduction. It has been consistently shown that AR mRNA levels increase dramatically following castration ,85-87 ann 2 days . Steinsaalf-life .et al. [et al. concluded that AR protein synthesis was involved in the mechanism for AR protein increase following testosterone administration.Consistent with these results, Mora et al.  showed tλ, and effective turnover rate of AR, λ using steady state analysis and useSince AR binding to androgen greatly increases its stability, androgen administration could increase the level of AR protein by increasing its stability. These results are collectively consistent with the notion that mRNA production and AR protein synthesis are both negatively regulated by androgens, but that a nonlinear decrease in the stability of AR occurs for very low androgen concentration. We have examined the production rate, This gives Rt, that along with CT:R and CD:R determines the AR production rateWhile such results imply that the AR level is not constant, castration is an extreme scenario , and since AR levels rebound shortly after androgen reintroduction ,89, it iwhere Ct, and the prostate cell count, P(t). There are two independent proliferation signals, one mediated by Ct and the other by S, and two similar death signals. In addition, the summed proliferation signal is attenuated by crowding and there is a background death rate. The shapes of these signals are collectively determined by the parameters θ1, θ2, φ1, φ2, ϕ, ω, n, m, and q. We have chosen values for these parameters based upon our determination of physiologic (and super- and sub-physiologic) parameter values for serum T and prostatic AR and the resulting Cts; they are reported in Table The growth of the prostate epithelium, in our model, is governed solely by the intraprostatic weighted AR:ligand complex concentration, S, is shown as a function of Ct in Figure Ct and S. It also shows the proliferation and death signals mediated by S as a function of Ct, since S is itself a function of Ct. Such curves emphasize that Ct is the ultimate mediator of all proliferation and death (besides crowding effects) in our model. These figures disregard the -σP crowding term as this varies depending upon prostate size. The qualitative form of these curves is preserved over most parameter space.Oxidative stress, Rt to a constant and all other variables to 0. Serum T concentration is prescribed, and the model is run to steady state, as shown in Figure α-reductase to produce DHT. For physiologic values of serum T and prostate AR, once steady state is reached most androgen is bound its receptor, and nearly all intraprostatic androgen is DHT. These dynamics are biologically expected.We briefly characterize the basic dynamics of the AR kinetics model. The time-dependent dynamics of the model are demonstrated by initially setting At steady state, most AR is bound at physiological levels of serum testosterone. For superphysiological serum testosterone, intraprostatic free DHT increases while AR:ligand complex concentration levels off; intraprostatic free T also increases modestly with high serum T. This qualitative pattern is observed regardless of the concentration of AR; an example is shown in Figure KD for DHT:AR binding was the same in carcinomas as in normal tissue, the actual on/off rates were much slower. In our model, changes in the on/off rates do not affect the steady state as long as the KDs remain constant, suggesting slower rates offer no benefit to carcinoma growth. However, our model does not take into account the effect of increased complex life-spans, and it may be useful to consider average life-span in future work.We examine how altering several central parameters in the kinetics model influences the steady state AR:ligand concentrations under different androgen environments. Lea and French  found thα-reductase: α, kcat, and KM and η (i.e. effective KM). All affect the steady state in a similar manner and change the ratio of DHT:total androgen in a nonlinear manner. The total amount of androgen in the prostate also decreases modestly in a nonlinear fashion when 5α-reductase is inhibited.We examine the parameters associated with 5R, T, and D. We find that for normal physiologic serum T, only βD, the degradation rate of DHT, has any significant effect on either total prostate androgen or the ratio of DHT:T; both decrease dramatically with increasing βD. At low serum T, βR, the degradation rate of free AR, is also important. Increasing βR significantly reduces total androgen, but does not affect the DHT:T ratio. Modifying the rate at which free T degrades, βT, has only minor effects.Finally, we investigate the degradation rates of et al. [βR) will augment the response to androgens by increasing the concentration of receptor bound androgen. Therefore, increasing the half-life of the free AR is one pathway through which cancers may overcome androgen blockade.Gregory et al.  found thRt is shown in Figure Rt for different serum T levels is given in Figure We now examine the model dynamics at the prostate growth level. The steady state prostate mass as a function of serum testosterone for different levels of Rt and then declines. This decline occurs because the growth signal induced directly by androgens saturates for high Rt (or Ct) while ROS levels increase. This causes the ROS proliferation signal to attenuate, while the death signal increases in magnitude. While at steady state dP/dt = 0, i.e. there is no net growth or death, the internal cell turnover rate varies with Rt and TS. This will affect the rate at which evolution can occur, as greater cell turnover will presumably increase the rate at which mutations accumulate. Lower androgen always increases the turnover rate in this model. Figure α-reductase inhibition.For higher levels of serum testosterone, prostate mass peaks at some level of We have performed a sensitivity analysis of all the growth model parameters and found that the qualitative shapes of the mass and turnover curves are preserved.Rt from 15 to 114 nM. That is, we set Q = 100 and i, to be 14 + i nM, i ∈ {1,2,...,100}. The total number of cells in each state is tracked through time, and the average Rt at all time steps is calculated. Figure Rt under different serum T levels. 5α-reductase inhibition, e.g., through finasteride treatment, also induces a low androgen environment, and Figure η (increasing effective KM).We use the state-transition model for the evolution of AR expression to determine how different androgen environments influence the prostate-wide evolution of AR expression. We choose our model to have 100 states representing Rt is actually slighter weaker. In comparison, low androgen environments (i.e. low serum T or low 5α-reductase) demonstrate the greatest selection for increased AR expression. Generally, the widespread appearance of strains expressing high levels of AR occurs abruptly and later in time than under normal androgen levels. While this selection occurs later in time, a higher average Rt is ultimately obtained.In general, our results indicate that physiologic serum T selects for modestly increased AR expression, as do all androgen environments. Therefore, the model predicts that even in healthy men, prostate epithelial cells will increase their AR expression and hence potential for malignancy with time. Under high serum T, selection for an elevated Rt is always preserved. The parameters θ1, θ2, and μ, which govern the level of ROS, have the greatest effect on the dynamics. Increasing μ, the background ROS level, causes low androgen to select for higher Rt earlier in time. Decreasing θ2, which implies higher ROS for relatively lower levels of Ct, also decreases the time to selection for Rt. Increasing θ1, which implies ROS is elevated under a lesser degree of androgen insufficiency, changes the dynamics most significantly. Large values of θ1 result in a two-phase pattern of selection for high Rt in low androgen environments: AR initially increases slowly, there is a sudden jump, and finally increases slowly again. This and a more typical pattern of AR evolution for low versus high androgen environments are shown in Figure Rt in all parameter space, and this selection becomes apparent later in time than under normal androgen for most parameter space.We have performed a sensitivity analysis to determine whether this behavior is preserved under different growth model parameter values. We have found that the prediction that low androgen selects for a greater final These results suggest that a low androgen environment may delay the development of a malignant phenotype, but result in a more malignant or therapy-resistant strain later in time. This result could also be interpreted to mean that low androgen reduces the overall incidence of cancer, as the expected time to the development of a malignant strain is increased, but those cancers that do arise may be more aggressive. This notion is consistent with the results of finasteride treatment in men .α-reductase inhibitors can increase selection for AR overexpression in prostate epithelium, while high androgens may weakly protect against AR overexpression. As a whole, the clinical literature and our theoretical results suggest the following hypothesis: that low intraprostatic AR:ligand concentrations, caused either by low serum testosterone or 5-α reductase inhibition, drive evolution towards decreased dependence upon androgens in prostate epithelium, therefore increasing the risk of cancer that is resistant to hormonal therapy. Moreover, such cancers may arise later in life than those cancers with lower sensitivity to androgens.We have developed a preliminary model of the binding kinetics of prostate androgens, their relationship to proliferation and death in the prostate epithelium, and the evolution of AR expression, and hence malignant potential, in different androgen environments. Thus, we have modeled prostate carcinogenesis at three distinct scales. At the receptor kinetics scale, we have described the essential interactions between T, DHT, and AR, and have fully parameterized this model using biological data. At the prostate organ level, we have taken both the direct and indirect ROS mediated effects of androgens on epithelial proliferation and apoptosis into account. At the highest scale, we have employed a state-transition model to study selection between epithelial strains expressing different levels of AR, and whose behavior is determined by the coupled AR kinetics and prostate growth model. Results using this evolutionary modeling framework have suggested that a low androgen environment caused either by low serum testosterone or clinical intervention with 5We must emphasize the preliminary nature of the model, and there are likely many ways to model the phenomenon that we are studying. We have used only one approach, and there is much room for improvement as well as future work. In particular, we have not (explicitly) modeled the essential interactions between stromal and epithelial cells that mediate prostate growth. Moreover, we have ignored the heterogenous architecture of the prostate. Not only do different prostate lobes display different responses to androgens ,67, but S increase with low androgen, but hypoxia is also likely spatially heterogeneous.We have not explicitly modeled the role of the prostate vasculature. Delivery of testosterone from the serum to the prostate is dependent upon prostate size and the state of the vasculature, both of which are themselves regulated by prostatic androgen, and vascular regression precedes prostate involution in castration models ,72. We hWhile much mechanistic and spatial complexity has been ignored, the relative simplicity of our model of tissue-level prostate growth is also a strength in that it allows several of the competing effects of androgens on prostate epithelium--i.e., oxidative stress, proliferation, apoptosis--to be subsumed into a single differential equation. Thus, the model includes the essential effects of androgens with minimum complexity, and the qualitative effects of androgens on the prostate epithelium can be explored in a minimal formal setting. However, we have not rigorously parametrized this level of the model, although its qualitative behavior is preserved over a wide range of parameter values. It follows that a weakness of the model framework, in its current form, is the difficulty in confronting the model with quantitative data other than in regards to its qualitative predictions.α-reductase activity , can decrease AR:ligand concentrations in a nonlinear manner.Our parametrization and investigation of the AR kinetics model has yielded some insight into AR dynamics. Our parametrization suggests that AR levels are constant under the physiologic range of serum androgens. We have found that increased stability of free AR has little effect on AR dynamics under normal androgen levels, but in a state of androgen deprivation, increased stability of the free receptor significantly increases intraprostatic AR:ligand concentrations, implying a role for such a modification in HR cancer, as seen in . Moreoveα-reductase is inhibited, the prostate epithelial cell mass is much lower. Thus, there is simply less substrate for selection to act upon when serum T is low. However, in such an environment the cell turnover rate is higher. Therefore, mutations may arise more rapidly and be selected for more quickly in a low androgen environment despite the lower cell count.In our model, when serum T is low or 5et al. [KM for growth varied up to 1,250-fold in [This, along with the results from our state-transition model, support the notion that low androgen levels can induce selection for phenotypes with increased AR expression. Such a phenotype may be considered to have a higher potential for malignancy or be "pre-malignant." In particular, such a phenotype is likely to be resistant to androgen ablation therapy, and a great deal of experimental and clinical literature supports the notion that low androgen levels induce treatment resistant cancers. Several studies by Labrie et al. ,91 on mo-fold in ) and thaet al. [A number of clinical studies have shown that low testosterone levels predict a poor response to androgen deprivation. In 1981, Adlercreutz et al.  found thet al. ,44,45,48et al. ,47. Furtet al. ,48.Daniell  reportedet al. found that not only did men with low testosterone have more aggressive cancers, but AR expression was also elevated in these patients [Many studies also suggest that low serum testosterone not only predicts a poor response to hormonal therapy, but also increases the risk of prostate cancer. Men with low free and total testosterone levels had a significantly higher rate of cancer than those with high serum testoserone . Schatzlpatients .On the other hand, several authors have concluded that low serum testosterone predicting more advanced disease or poorer response to therapy simply reflects the poorer health of such patients rather than indicating any causal role for testosterone in cancer development ,41. HoweFollowing the results of the PCPT, which showed an overall reduction in cancer incidence for those taking finasteride but an increase in high-grade tumors , great cα reductase inhibition on selection for AR overexpression is similar to the effect of low serum T. That is, AR overexpression is selected for more strongly, but the expected time to such overexpression being observed is greater than in a normal androgen environment. Therefore, we expect 5-α reductase inhibition to similarly increase risk for therapy-resistant and/or aggressive prostate cancer. Indeed, as early as 1993, Martel et al. [α-reductase inhibitors in the treatment of BPH could induce selection for therapy resistant cancer cells.In our model, the effect of 5-l et al.  expresseRt remains low for a long time, but increases dramatically late in time. Therefore, in a relatively short temporal window overall cancer incidence may be reduced, but a minority of patients will cross the threshold for increased AR expression and experience a high-grade tumor. In light of the clinical data on low testosterone and cancer risk, and our own results suggesting that the temporal scale for the incidence of aggressive cancer under 5α-reductase inhibition is not the same as that for overall cancer incidence under normal androgen environments, long-term follow-up of patients being treated with finasteride is warranted, and the response to hormonal therapy among patients who do experience prostate cancer should be studied.Perhaps low serum T consistently predicts both increased cancer incidence and aggressiveness because in men with lifelong low testosterone the temporal window where a normal or high androgen environment results in greater AR expression 1. The idea proposed in this article that there are "sweet spots" in the concentrations of hormones and in stages of development of the tumor that guide tumorigenesis and lead to therapy resistance is an important concept towards personalize cancer treatment. It reminds me of the analogy of the pepper shaker and the steak: a little pepper makes the steak taste better but a lot of pepper makes the steak a lot worse.I would suggest rewriting this sentence for sake of clarity "Most cancers are more aggressive following HR recurrence, there are no effective treatments for such cancers, and average survival following progression does not exceed 15 months ." I undeAuthors' response: Yes, that is exactly what was meant by this sentence. 2. Please review these two sentences; they seem to mean the same thing: "AR amplification occurs in perhaps 30% of recurring HR tumors . HoweverAuthors' response: Here we are pointing out that while AR gene amplification does occur and is not an insignificant contributor to HR tumor recurrence, such a genetic alteration does not occur in the majority of HR tumors. You are correct that the second sentence is somewhat redundant, but we have left it in to emphasize that this is not the dominant pathway by which HR recurrence occurs .3. Please clarify how this paragraph relate to previous observation, it seems contradictory that the upregulation of AR expression is HR tumors but is also the most important pathway: "Upregulation of the AR protein is perhaps the single most important pathway by which cancers achieve androgen independence. Chen et al.  found thAuthors' response: In the previous paragraph, we are pointing out that amplification of the AR gene occurs in a minority of HR tumors. Increased levels of the AR protein itself, whether achieved through gene amplification or by other pathways, seems to be the most important (or at least most common) pathway to HR tumor recurrence in the majority of tumors.4. Could you please give more details on this trial? The subjects had any signal of prostate tumor or the idea is that every man would submit to this treatment after a certain age? "In the Prostate Cancer Prevention Trial (PCPT), finasteride use reduced overall prostate cancer incidence, but increased the risk of high-grade cancer over 7 years ."Authors' response: The PCPT was a double-blind RCT that assigned men with no history of prostate cancer to receive finasteride (5 mg) or a placebo daily for 7 years. Biopsy was performed on the basis of serum PSA or abnormal digital rectal exam. At the end of the 7-year study period, all men who consented received a biopsy. Finasteride use reduced overall prostate cancer incidence by 24%, but increased the risk of high-grade cancer: 37% of cancers were high-grade in the treatment group versus 22% in the placebo group. These details are now included in the text.5. Could you please explain the relationship between a smaller prostate and the probability that a biopsy would detect cancer? We could imagine that smaller prostates would pass the rectal touch exam and thus not be biopsied? "Finastide significantly reduced the prostate size in those treated, and reduced prostate size can increase the probability of cancer detection in biopsy samples, so a detection bias could explain the increased rate of high-grade cancer."Authors' response: What is being discussed here is not the relationship between the prostate size and the probability of biopsy based on DRE findings .Rather, it has been argued that given a biopsy occurs, the probability that tumor tissue is present in the biopsied tissue sample will depend on the absolute size of the prostate. If more normal epithelium is present, as in the case of placebo-treated men, the probability that tumor tissue is detected decreases. Since finasteride treatment reduces the prostate volume, a tumor of equal size is more likely to be detected in biopsy samples from a finasteride-treated man than a placebo-treated man. This has been (hopefully) clarified in the text.α [6. Please elaborate on how hypoxia may induce increase in ROS. One of the explanations for limited effect of radiation therapy in hypoxic areas of tumors in the low levels of reactive oxygen species, supposedly due to the lack of oxygen. "Hypoxia impairs aerobic respiration, and mitochondrial ROS is required for stabilization of HIF-1α . Thus, iAuthors' response: Radiation exerts its biological effects primarily through the ionization of water:These and other subsequently generated radical species can react with O2 to form reactive oxygen species such as the superoxide anion and hydrogen peroxide. These species initiate further radical reactions that damage the DNA. Therefore, the presence of molecular oxygen in tumor tissue, but not necessarily the presence of pre-existing ROS, is an important determinant of the efficacy of radiation therapy.In a hypoxic environment the electron transport chain is impaired and oxygen is less likely to be fully reduced, resulting in partial oxygen reduction and ROS generation. Such mitochondria generated ROS stabilizes HIF-1α and thus activates the cellular response to hypoxia. We have modified this section slightly for clarity.α reductase inhibition, drive evolution towards elevated sensitivity to androgens in prostate epithelium, therefore increasing the risk of cancer that is resistant to hormonal therapy" might be better understood if the words "elevated sensitivity" were replaced by "smaller dependency."7. The sentence "that low intraprostatic AR:ligand concentrations, caused either by low serum testosterone or 5-Authors' response: We have replaced "elevated sensitivity to" by "decreased dependence upon."Dr. Marek Kimmelet al. 2007 from Houtsmuller's laboratory [In the transcription factor literature, there is a significant number of papers concerning spatial organization of AR activity within the nucleus, leading to effects such as hyperspeckling (see the attached paper by van Royen boratory ). Are thAuthors' response: In this paper, van Royen et al. present a model for AR/DNA/cofacter interaction that is modulated by binding between the N terminal and C terminal domains (N/C interactions) of the AR. Such binding appears to prevent cofactor recruitment, and occurs upon hormone ligand binding. Following ligand binding, the AR translocates to the nucleus, where it is highly mobile. Transient binding to the DNA terminates N/C interactions. This allows the C-terminal ligand binding domain (LBD) to interact with cofactors containing the FxxLF motif, leading to altered transcriptional activity.It is likely that cancer-specific alterations of the AR that affect the spatial kinetics of DNA binding and cofactor recruitment play a role in prostate cancer progression and its response to treatment. For example, changes in N/C interaction might alter co-factor recruitment within the nucleus, and altered co-factor activity may be associated with HR recurrence. Farla et al. [showed that while both AR agonists and antagonists cause translocation of the AR:ligand complex to the nucleus, only agonists caused DNA binding. Mutations in the AR can cause antagonists to act as agonists, and these may act by altering N/C interaction and cofactor recruitment following binding. Farla et al. established that DNA binding kinetics for mutant ARs bound to anti-androgens were similar to the DNA binding kinetics for wild-type ARs bound to agonists. Therefore, modification of DNA binding in the spatial compartment in response to non-canonical ligands is likely one pathway by which prostate cancers become hormone refractory. van Royen et al. also found that speckling overlaps with, but is not perfectly correlated with, transcriptional activity. However, speckling does not occur without DNA binding, so the dynamics of speckling are likely a direct result of DNA binding kinetics and subsequent interactions with co-factors, etc.a et al. showed thIn any case, we would argue that any alteration in the AR that affects the transcriptional response to AR:ligand binding must necessarily affect the spatial dynamics of AR activity in the nucleus. At the phenomenological level, the exact molecular mechanisms by which AR activity is changed are not as important as how the androgen environment selects for altered AR activity in general.


In clinical medicine there may be times when clinical conditions manifest differently both when they present individually or concomitantly. Such scenarios warrant a broader differential diagnosis with thorough investigations. We present one such case of a patient of Bell's palsy with unexplained eye pain on the ipsilateral side. The patient had a chronic retinal detachment which became worse due to the concomitant Bell's palsy. A principle of Osler is that a constellation of symptoms is caused by one disease process. There are occasionally presentations that do not adhere to this principle. We present a classic presentation of a Bell's palsy with unexplained eye pain on the ipsilateral side. The patient had a chronic retinal detachment which became worse concomitantly with the Bell's palsy. While the two entities are unrelated it did create a large differential diagnosis requiring an extensive evaluation. We present the case report and a review of the diagnosis and management of Bell's palsy.A 47-year-old African-American man walked into the Emergency Department complaining of right-sided facial numbness and weakness. Before bed the previous night, the patient noted that fluid dripped out of the right side of his mouth while drinking. He then reawakened in the morning to right-sided facial droop, inability to close right eye and numbness to the right side of his face. He denied any numbness, tingling, or weakness in his extremities. On review of systems, he noted slight pain behind his right eye intermittently for the past few days and noted that he had developed a floater in his right eye 5 days ago. He denied recent cold sores, ear discharge, or recent trauma; however, he noted flu-like symptoms about 1-2 weeks ago. He also denied any changes in hearing or ear pain. The patient noted no previous medical history, however had not seen a doctor in more than 20 years. He denied any prior surgeries. He is a smoker but denied alcohol or illicit drug use.On exam, the patient was in no acute distress and in no obvious discomfort. He was hypertensive with an initial blood pressure of 182/108. His speech was slightly slurred. He was alert and oriented times four. His skin was warm and dry with no apparent rashes or lesions. No nystagmus was noted. He demonstrated an inability to close his eyelid as well as generalized weakness of his right face including his forehead. His pupils were 3 mm, equal, round, and reactive. Extraocular movements were intact. Corrected visual acuity was 20/30 OD, OS, and OU. The fundus was not visualized on the right while the left appeared normal. No ulcerations were noted in his ears. The remainder of his exam including a thorough neurologic exam was unremarkable.All diagnostic tests were essentially normal, including electrocardiogram and labs. Magnetic resonance imaging and magnetic resonance angiography of head and neck demonstrated no acute findings. Vascular tortuosity was noted, consistent with longstanding hypertension.Initial differential diagnosis for this patient included: stroke, Bell's palsy, vascular disease including carotid dissection, retinal detachment, herpes zoster ophthalmicus, middle ear lesions including Ramsey-Hunt syndrome, Human immunodeficiency virus, Lyme disease, Guillain-Barré syndrome, ocular lymphoma and vitreous hemorrhage, or inflammation. However, none of these diagnoses could explain the patient's facial paralysis and eye pain. Once imaging studies revealed no acute pathology and ophthalmologic testing revealed lattice degeneration and retinal detachment, it became clear that this patient had Bell's palsy with an incidental, likely chronic but only recently symptomatic retinal detachment. Upon thorough review of the literature, no cases were noted suggesting any link between the presentations of these separate processes. Once this patient's ocular symptoms were accounted for by the retinal detachment, his neurologic symptoms represented a classic presentation of seventh nerve palsy. His course was significant for complete recovery of facial nerve function on acyclovir and prednisone in addition to successful surgical correction of his retinal detachment.Bell's palsy is a common neurologic disorder often inciting significant concern in patients and triggering stroke alerts in emergency departments. It is the most common cause of acute facial paralysis. According to one study, 68% of acute peripheral facial palsy were classified as Bell's palsy. Incidenc24Bell's palsy is believed to be caused by inflammation of the facial nerve at the geniculate ganglion, which lead to compression and possible ischemia and demyelination. Bell's pAlthough Bell's palsy is often referred to as idiopathic, the cause can be infectious in nature. Herpes simplex virus activation has become widely accepted as the likely cause of Bell's palsy in most cases. Polymerase chain reaction DNA testing now verifies the notion of axonal spread and multiplication of a reactivated neuron-tropic virus leading to inflammation, demyelination, and palsy.8 Other iPatients with Bell's palsy complain of sudden facial weakness (over the course of a few hours), difficulty with articulation, inability to keep an eye closed, or inability to keep food or drink in the mouth on one side. The seventh cranial nerve also serves other functions; the patient may have variable degrees of dry eye, metallic taste of the mouth, and facial pain commonly around the ear. Depending upon the site of the lesion, there may be associated impairment of taste, lacrimation, or hyperacusis. It is not uncommon for patients to have ipsilateral facial sensory impairment suggestive of ipsilateral trigeminal neuropathy such as in this particular patient. Such ipsilateral sensory loss in the setting of Bell's palsy has usually been attributed not to neuropathy, but to abnormal perception based on droopy facial muscles, skin, and associated tissue.The examination is notable for unilateral weakness of the face involving the forehead. The facial creases and nasolabial fold disappear, the forehead unfurrows, and the corner of the mouth droops. The eyelids will not close and lower lid sags. On attempted closure of the eyelid, the eye rolls upward (Bell's phenomenon). Tear proThe diagnosis of Bell's palsy is essentially one of exclusion. While as previously described it is the most common cause of acute facial paralysis, its presentation is similar to potentially devastating events such as cerebrovascular accident (CVA) and tumor. No readily available laboratory test or imaging can verify the diagnosis of Bell's palsy. Key points to differentiate Bell's palsy from other etiologies include a rapid onset and progressive course, diffuse facial nerve involvement, normal reflexes, lack of vesicular lesions. Laboratory testing is usually not indicated unless the patient is at high risk for Lyme disease. Imaging is often warranted if other diagnoses are being considered, especially in the setting of an atypical presentation including insidious onset and forehead sparing. ElectrodTreatment for Bell's palsy is etiology-driven if a specific case is identified. Most patients with Bell's palsy recover without treatment 71% achieve complete recovery, 84% achieve near normal function. CommonlyControversy remains regarding the therapeutic effectiveness of steroids and acyclovir. According to the Cochrane Reviews, the available evidence from randomized controlled trials does not show significant benefit from treating Bell's palsy with steroids alone. Upon comTreatment with antivirals seems logical in Bell's palsy as the etiology is often viral. The American Academy of Neurology considers acyclovir safe and possibly effective in improving functional outcomes. However,Other proposed treatments with little evidence to support them include Methylcobalamin, hyperbaric oxygen, facial retraining, botulinum toxin, transcutaneous electrical stimulation, and acupuncture.Sir Arthur Conan Doyle wrote; “It is an old maxim of mine that when you have excluded the impossible, whatever remains, however improbable, must be the truth.” This unu


Studies in heterologous cell systems have shown that this receptor is expressed in intracellular compartments. Other studies show that dimerization with other ARs promotes the cell surface expression of the α1D-AR. To assess the cellular localization in vascular smooth muscle cells, we developed an adenoviral vector for the efficient expression of a GFP labeled α1D-AR. We also measured cellular localization with immunocytochemistry. Intracellular calcium levels, measurement of reactive oxygen species and contraction of the rat aorta were used as measures of functional activity.The cellular localization of the α1D-AR was expressed in intracellular compartments in human aortic smooth muscle cells. The intracellular localization of the α1D-AR was also demonstrated with immunocytochemistry using an α1D-AR specific antibody. RT-PCR analysis detected mRNA transcripts corresponding to the α1A-α1B- and α1D-ARs in these aortic smooth muscle cells. Therefore, the presence of the other α1-ARs, and the potential for dimerization with these receptors, does not alter the intracellular expression of the α1D-AR. Despite the predominant intracellular localization in vascular smooth muscle cells, the α1D-AR remained signaling competent and mediated the phenylephrine-induced increases in intracellular calcium. The α1D-AR also was coupled to the generation of reactive oxygen species in smooth muscle cells. There is evidence from heterologous systems that the α1D-AR heterodimerizes with the β2-AR and that desensitization of the β2-AR results in α1D-AR desensitization. In the rat aorta, desensitization of the β2-AR had no effect on contractile responses mediated by the α1D-AR.The adenovirally expressed α1D-AR with other ARs does not alter the cellular expression or functional response characteristics of the α1D-AR.Our results suggest that the dimerization of the α Three αcterized ,4. These failure -8.1B-AR is localized on the cell surface, as expected for a GPCR, while the α1A-AR is localized not only on the cell but also in intracellular compartments [1D-AR is localized intracellularly [It is well known that the localization and trafficking properties of a receptor can modulate its physiological function . Resultsartments ,11. In cllularly . These rllularly .1D-AR interacts with the α-AR interacts with the α1B-AR and the β2-AR [1D-AR. This has lead to the suggestion that these receptors are capable of heterodimerization [1D-AR. We show that despite the presence of the other α1-AR family members, the α1D-AR is expressed mainly in intracellular compartments. We further show that while receptor dimerization may occur, it does not appear to alter the functional properties of the α1D-AR.In recent years, the concept of receptor dimerization has brought a new perspective to GPCR function -16. Prevhe β2-AR  resultinrization -20. Thes1D-AR. Infection of aortic smooth muscle cells with virus expressing the α1D-AR/GFP resulted in approximately 80% receptor transfectional efficiency (not shown) demonstrating that adenovirus can be useful in cells that have been traditionally difficult to transfect with the α1-ARs. Following viral infection, the α1D-AR/GFP was detected in intracellular compartments of aortic smooth muscle cells  the α1D-AR is nonetheless expressed intracellularly. Further, the data argue that while dimerization may occur in smooth muscle cells, it does not alter the localization of the α1D-AR.It is possible, however, that in these cultured smooth muscle cells the αs Figure . As has 1-AR blocker prazosin or 30 nM of the highly selective α1D-AR antagonist BMY 7378. To substantiate the selectivity of this dose of BMY 7378 we measured phenylephrine-induced increases in intracellular calcium levels in fibroblasts stably transfected with either the α1A-AR, α1B-AR or the α1D-AR. Despite pretreatment with 30 nM BMY 7378, phenylephrine maintained the ability to promote increases in intracellular calcium in the α1A-AR or α1B-AR expressing lines of fibroblasts . Thereft Figure . However1-ARs are a novel and efficient tool to investigate properties of these receptors in native cells. These vectors were used to show that in human smooth muscle cells expressing all three α1-ARs, the α1D-AR is localized in intracellular compartments. Therefore, despite recent reports of heterodimerization between the α1-ARs, in a human vascular smooth muscle cell line, the α1D-AR is still expressed intracellularly. Indeed none of the data we obtained in this work support the idea of α1D-AR heterodimerization. Using three independent measures  we also could not detect any evidence of altered pharmacologic properties of the α1D-AR.In summary, adenoviral vectors expressing the α1-AR subtypes were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum and a 1% antibiotic/antimycotic cocktail . All cells were grown in T75 flasks in a 37°C cell culture incubator with a humidified atmosphere (95% air and 5% CO2) and were fed every 2 to 3 days. After reaching confluence the cells were plated on plain untreated coverslips in 35 mm tissue culture dishes.Human aortic smooth muscle cells were obtained from Cascade Biologics  and grown in Medium 231 supplemented with smooth muscle growth supplement until they become confluent . Stably transfected Rat 1 fibroblast lines expressing each of the α1D-AR coupled to the green fluorescent protein (α1D-AR/GFP) was provided by Dr. Gozoh Tsujimoto [EcoRI and XbaI enzymes and cloned into the pCI expression vector . pCI was digested with BglII and ClaI. This fragment included the CMV I.E. promoter, the α1D-AR/GFP and the SV40 late poly (A). The BglII and ClaI fragment was cloned into the adenovirus recombination vector pAdLink. pAdLink and the wild type adenovirus vector, dl327, were linearized with NheI and ClaI respectively. Homologous recombination occurred by co-transfecting linearized pAdLink and the wild type adenovirus vector into HEK 293 cells. Positive plaques appeared 10 to 14 days after recombination and were then amplified. Plaques were purified by serial dilutions of a positive plaque  in 96 well plates using HEK 293 cells. After plaque purification, samples of viral DNA were analyzed for wild type virus contamination by PCR [A vector expressing the human αsujimoto ,24. Thisn by PCR . Once a or Rat 1 fibroblasts were grown on glass coverslips. Two hours prior to infection, cells were placed in serum free medium and infected with adenovirus. Twenty four hours after infection the medium was changed and the virus free incubation was allowed to proceed for an additional 24 hours. Cells expressing the GFP labeled α1D-AR were fixed with 3.7% Formaldehyde in PBS for 10 mins. Cells were then mounted on slides with Vectashield . Cells were visualized using a Leica TCS SP 5 AOBS confocal microscope with a Plan-Apo 64X oil immersion objective lens  using Leica TCS NT version 2.5 software. Images were transferred to a computer for reduction with Adobe Photoshop version 6.0 .Human aortic smooth muscle cells 1-ARs, diluted 1:100 in 1% BSA in PBS, was added and incubated overnight at 4°C. Following this incubation, the cells were washed with .05% BSA in PBS and a Texas Red secondary antibody , diluted 1:500 in PBS, was added and incubated in the dark at room temperature for 1 hr. Cells were washed with PBS and mounted on glass slides with Vectashield . Cells were visualized with a confocal microscope as described above.Human aortic smooth muscle cells or grown on glass cover slips, were washed in PBS and fixed with 3.7% Formaldehyde in PBS for 10 min. Cells were then washed with .05% BSA in PBS and permeabilized with 0.1% Triton in PBS for 5 min. After permeabilization, the cells were washed and blocked with 10% lamb serum for 1 hour at room temperature. After washing polyclonal antibodies  against each of the α20 primer . After heating at 85°C for 5 min. to terminate the reaction, cDNA samples were stored at -20°C until used. Negative controls for the presence of genomic DNA were performed by replacing the reverse transcriptase enzyme with Taq DNA polymerase.Total RNA from HASMCs was isolated and purified with the ChargeSwitch Total RNA kit from Invitrogen , and 0.5 μg samples were reverse transcribed at 45°C for one hour using Cloned Avian Myeloblastosis Virus (AMV) Reverse Transcriptase and Oligo(dT)et al. [1A, 5'-ATGCTCCAGCCAAGAGTTCA-3'  and 5'-TCCAAGAAGAGCTGGCCTTC-3' ; α1B, 5'-CTGTGCGCCATCTCCATCGATCGCTAC-3'  and 5'-ATGAAGAAGGGTAGCCAGCACAAGATGAA-3' ; α1D, 5'-CTCTGCACCATCTCCGTGGACCGGTAC-3'  and 5'-AAAGAAGAAAGGGAACCAGCAGAGCACGAA-3' . The receptor specific primers target sequences within the third intracellular loop (sense) and the carboxy terminus (antisense). Primers for β-actin were included as a positive control . The predicted sizes of the amplified human β-actin, α1A-, α1B-, and α1D-AR PCR products were 353, 502, 530, and 540 bp, respectively.For PCR, primers were synthesized by Invitrogen based on those used by Esbenshade et al.  to detecTaq DNA polymerase (Invitrogen) in a PCR Express  thermal cycler. The amplification reactions, repeated for 35 cycles, consisted of denaturation at 94°C for 1 minute, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute. PCR products were run on a 1.4% agarose gel.PCR was carried out with Platinum Human aortic smooth muscle cells were loaded for 1 hour with 5:M Calcium Green ™ 1 AM . In certain experiments, Rat 1 fibroblasts were also loaded with the Calcium Green. Cells were then washed twice with serum containing medium and visualized with an inverted microscope with a Xe arc lamp with a Plan-Apo 60X oil immersion objective and an excitation filter of 480/15 nm and an emission filter of 535/20 nm. Images were taken using a CoolSnap HQ camera. Indicator dye-loaded cells underwent several drug treatments. Human aortic smooth muscle cells were pre-treated with vehicle, 1 nM prazosin or 30 nM BMY 7378 for 20 minutes, followed by 25 :M phenylephrine. Stably transfected fibroblasts were challenged with 10 :M phenylephrine. A higher phenylephrine concentration was required in smooth muscle to observe an equivalent increase in the calcium signal. Images from calcium measurements were processed using Metamorph software . In the analysis, the nucleus was masked leaving the cytoplasm. The mean intensity of the cytoplasm was then taken for each cell. Images were prepared using Adobe Photoshop version 6.0 . lmaging data were analyzed by one-way analysis of variance with Tukey's post-hoc test using GraphPad Prism version 3.00 for Windows . In all figures, the data are expressed as the mean and standard error of the mean (S.E.). A value of P < 0.05 was considered statistically significant.The generation of reactive oxygen species was measured using Mitotracker ROS  diluted in DMSO. Human aortic smooth muscle cells, attached to glass coverslips, were incubated for 20 min at 37° with 5 nM Mitotracker ROS diluted in Serum Free Medium. Cells were washed twice with medium and fresh medium applied. After this time 10 μM phenylephrine was added and incubated with the cells for 20 min. Preliminary studies established this time as that optimal to record a significant increase in ROS levels. In certain experiments, cells were pretreated with 1 nM prazosin or 30 nM BMY 7378 prior to the addition of phenylephrine. After agonist incubation, the cells were fixed with 1.0% formaldehyde in PBS for 10 min and washed with PBS. Cells were mounted on slides with Vectashield . Cells were imaged by confocal microscopy as described above. Images were processed using Metamorph software . Images were prepared using Adobe Photoshop version 6.0 . lmaging data were analyzed by one-way analysis of variance with Tukey's post-hoc test using GraphPad Prism version 3.00 for Windows . In all figures, the data are expressed as the mean and standard error of the mean (S.E.). A value of P < 0.05 was considered statistically significant.2-AR agonist or a vehicle control. After this period, rings were placed in the tissue baths for the assessment of contractile activity. The water-jacketed muscle baths were filled with physiologic saline solution maintained at 37°C with constant oxygenation  and under a passive force of 2.0 grams. Previous studies have shown that this passive force gives optimal responses. Aortic rings were contracted with 25 mM KCl and the ability of increasing amounts of albuterol to induce aortic relaxation was measured. Phenylephrine-dose response curves were also generated in a separate set of aortic rings treated with albuterol. Changes in the force generation were recorded using force displacement transducers  interfaced to a Dell computer. Data were retrieved using PolyVIEW version 2.5 and analyzed using GraphPad Prism version 3.00 for Windows .All animal protocols were reviewed and approved by the University of Kentucky Institutional Animal Care and Use Committee. Isolated blood vessels were prepared by techniques routinely used in our laboratory ,22,27,28AR: Adrenergic receptor; GPCR: G-Protein-Coupled Receptor; HASMC: Human aortic smooth muscle cell; GFP: Green fluorescent protein.MLG performed or assisted in many of the studies in the manuscript. Specifically, figures


Metabolic changes inducedby hyperglycemia lead to dysregulation of cytokine control.Moreover, their regulatory roles in nerve degeneration and regeneration may potentially be utilized for the preventionand/or therapy of diabetic neuropathy.Diabetic neuropathy develops as a result of hyperglycemia-induced local metabolic and microvascularchanges in both type I and type II diabetes mellitus. Diabeticneuropathy shows slower impulse conduction, axonal degeneration,and impaired regeneration. Diabetic neuropathyaffects peripheral, central, and visceral sensorimotorand motor nerves, causing improper locomotor and visceralorgan dysfunctions. The pathogenesis of diabetic neuropathyis complex and involves multiple pathways. Lackof success in preventing neuropathy, even with successfultreatment of hyperglycemia, suggests the presence of earlymediators between hyperglycemia-induced metabolic andenzymatic changes and functional and structural propertiesof Schwann cells (SCs) and axons. It is feasible thatonce activated, such mediators can act independently of theinitial metabolic stimulus to modulate SC-axonal communication.Neuropoietic cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6), leukemia inhibitory factor (LIF),ciliary neurotrophic factor (CNTF), tumor necrosis factoralpha (TNF-


Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes α5β1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with α5β1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active α5β1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with α5β1, and the associated motor myosin VI (Myo6) support active α5β1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active α5β1. Nrp1 modulation of α5β1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165  and semaphorin 3A (SEMA3A). Nevertheless,  The vascular system is a hierarchical network of blood vessels lined by endothelial cells that, by means of the transmembrane integrin proteins, bind to the surrounding proteinaceous extracellular matrix (ECM). Integrins are required for proper cardiovascular development and exist in bent (inactive) and extended (active) shapes that are correspondingly unable and able to attach to the ECM. Extracellular guidance cues, such as vascular endothelial growth factor and semaphorins, bind the transmembrane protein neuropilin-1 (Nrp1) and then activate biochemical signals that, respectively, activate or inactivate endothelial integrins. Here, we show that Nrp1, via its short cytoplasmic domain and independently of vascular endothelial growth factor and semaphorins, specifically promotes endothelial cell attachment to the ECM protein fibronectin, which is known to be crucial for vascular development. Notably, Nrp1 favors cell adhesion by associating with fibronectin-binding integrins and promoting the fast vesicular traffic of their extended form back and forth from the endothelial cell-to-ECM contacts. Binding of the Nrp1 cytoplasmic domain with the adaptor protein GIPC1, which in turn associates with proteins required for integrin internalization and vesicle motility, is required as well. It is likely that such an integrin treadmill could act as a major regulator of cell adhesion in general. The transmembrane protein neuropilin-1 promotes endothelial cell attachment to the extracellular matrix by enhancing active integrin treadmilling at cell-adhesion sites. In vertebrates, the development of a hierarchically organized and functional vascular tree relies on the dynamic interaction of endothelial cells (ECs) with the surrounding extracellular matrix (ECM), which is mediated by heterodimeric αβ integrin adhesive receptors . During The biological activities of integrins depend on the dynamic regulation of their adhesive function in space and time. In cells, integrins exist in different conformations that determine their affinities for ECM proteins  and are The extracellular region of Nrp1 contains two repeated complement-binding domains , two coagulation-factor-like domains (b1-b2 domains), and a juxtamembrane meprin/A5/μ-phosphatase  homology domain. The Nrp1 intracellular region is only 50 amino acids in length, and its function is poorly characterized . ThroughNrp1 null mice display an embryonic lethal phenotype, characterized by dramatic vascular defects ascribed to impaired angiogenic sprouting [120/120VEGF mice) [Nrp1–/– embryos die in utero by 13.5 days postcopulation [120/120VEGF pups are recovered at birth at a normal Mendelian frequency [Nrp1 null mutants, i.e., the severe impairment of neural tube vascularization [120/120VEGF mouse embryos [Nrp1 null mice [Sema3a null mice is significantly influenced by the genetic background [Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in vascular cells, such as adhesion to the ECM [.prouting , branchiprouting , or arteprouting  that is 20 mice)  or Sema320 mice) . Indeed,pulation , VEGF120requency . Moreoverization , is not  embryos . In addiull mice ,24, the ckground ,28–30. T the ECM ,32. HowehNrp1) or control nontargeting siRNA (siCtl). Twenty-four hours after the second transfection, Western blot analysis revealed that, in comparison with control cells, Nrp1 protein, but neither β-tubulin nor the Nrp1 interactor GAIP interacting protein C terminus, member 1 (GIPC1), was successfully silenced in sihNrp1 ECs . Parenthetically, Nrp1 has been found to be expressed at higher levels in arteries than in veins . EndotheNrp1 ECs A.Next, we investigated the effect of Nrp1 silencing on EC adhesion to different ECM proteins. Fibronectin, vitronectin (VN), and type I collagen (COLL-I) are typical constituents of the provisional angiogenic ECM , whereashNrp1 ECs were impaired in their ability to incorporate endogenous sFN into a dense fibrillar network 3 h after plating  to rescue the adhesion and fibrillogenesis defects of sihNrp1 ECs -tagged full-length (mNrp1) and deletion mutants of murine Nrp1 (mNrp1dSEA) or the whole cytoplasmic domain (mNrp1dCy). The sihNrp1 pool did not target any of the mNrp1 constructs, and immunoprecipitation experiments on membrane-biotinylated cell monolayers revealed that all three transmembrane proteins were efficiently exposed on the cell surface -tagged α5 integrin subunit (α5-GFP) [We then examined whether in ECs Nrp1 could interact physically with α5β1 integrin. Lysates from ECs adhering on endogenous ECM were immunoprecipitated with an antibody (Ab) recognizing the FN receptor α5β1 and then blotted with anti-Nrp1 Ab. Nrp1 coimmunoprecipitated with α5β1, and blotting Nrp1 immunoprecipitates with anti-α5β1-integrin Ab further confirmed the association between endogenous n in ECs C. To bet(α5-GFP)  were immmNrp1 construct (mNrp1-mRFP) that was then cotransfected with α5-GFP in ECs. Fluorescent confocal microscopy showed that at the plasma membrane of ECs adhering on FN mNrp1-mRFP was enriched in close proximity to, or even tightly intermingled with, α5-GFP-containing adhesion sites -tagged on sites A, arrowsvinculin B or α5β1vinculin C at eithvinculin C, arrowsvinculin C, indicamNrp1 tagged with the fluorescent protein Cherry, an improved version of mRFP (mNrp1-Cherry). Total internal reflection fluorescence (TIRF) illumination [mNrp1-Cherry, indicating that FRET, and thus a close physical interaction, was occurring between α5β1 and Nrp1 at adhesion sites with an 11.5% FRET efficiency  in live NIH 3T3 cells transfected with α5-GFP alone or cotransfected with α5-GFP and mination  was usedmination  and was ficiency .Taken together, these data indicate that in living cells Nrp1 physically associates with α5β1 at or near sites of cell–ECM contact and that this interaction is likely maintained following internalization of the complex.hNrp1 ECs  integrin at the cell surface ,6. We foNrp1 ECs A, thus shNrp1 cells  α5-GFP (α5-PA-GFP) probe that we had previously used to monitor α5β1 trafficking in human ovarian carcinoma A2780 cells . However1-Cherry , arrows.vesicles  that movvesicles , with thvesicles .mNrp1-Cherry vesicles and not at the plasma membrane above and below them.Having established that α5β1 integrin and Nrp1 are cointernalized at fibrillar adhesions, we wished to determine whether the integrin was then recycled from Nrp1-positive vesicles back to the plasma membrane. To address this, we aimed a pulse of 405-nm laser light at a “single point” corresponding to Nrp1-positive vesicles, leading to the immediate photoactivation of α5-PA-GFP integrin largely within the confines of these structures . During Taken together, these data indicate that α5β1 integrin and Nrp1 are cointernalized into intracellular vesicles, which are then rapidly returned or recycled to the plasma membrane. Interestingly, both the internalization and the recycling of Nrp1-associated α5β1 integrin occur at the site of adhesion to the ECM.mNrp1-mRFP and Rab5-GFP, which were incubated with the α5β1 integrin activation reporter mAb SNAKA51 for 30 min at 4 °C and then at 37 °C for different time points. Fluorescent confocal analysis indicated that, after 1–3 min of internalization at 37 °C, Nrp1 and active α5β1 integrin colocalized in early Rab5-positive vesicles near the EC plasma membrane  that has also been involved in endocytosis , we conshampered B, indicahNrp1 and sihGIPC1 ECs, the endogenous FN fibrillogenesis defect seen in sihMyo6 ECs was due to an inhibition of FN1 gene transcription mRNA  [–/–Nrp1 mice could be due at least in part to the disruption of a VEGF-A165/SEMA3A-independent Nrp1 function. α5β1 Integrin and its ligand FN are key players in vascular development [Defects of developing blood vessels caused by  in mice ,24 are d in mice  or VEGF-20 mice) . Further20 mice) ,59. TherhNrp1 was simultaneously knocked down by RNAi, we showed that EC adhesion to FN and polymerization of endogenous sFN into fibrils depend on the cytoplasmic domain of Nrp1, the C-terminal SEA motif representing the minimal sequence required to exert these functions. Importantly, as already shown for SEMA3A-elicited growth cone collapse in neurons [In rescue experiments, where we reintroduced full-length and mutant murine Nrp1 constructs in human ECs in which endogenous  neurons , we foun neurons . This mo neurons , selectiIt is known that conformational activation of cell-surface integrins supports cell adhesion and spreading, whereas transition of integrins toward an inactive bent conformation causes cell de-adhesion and rounding up ,60,61. HTaken together, our data indicate that, during EC adhesion and spreading on FN, Nrp1, through its extracellular domain, transiently interacts with active α5β1 integrin at adhesive sites and, via its cytoplasmic association with GIPC1, enhances the early endocytosis and the ensuing recycling of active α5β1 integrin to newly forming adhesion sites A. It is hMyo6 ECs is likely due to the lack of the transcriptional activity displayed by Myo6 in the nucleus [FN1 gene transcription  and rabbit polyclonal anti-β-tubulin (H-235) were from Santa Cruz Biotechnology. Mouse monoclonal anti-human-Nrp1 (MAB 3870) was from R&D Systems. Mouse monoclonal anti-FN (MAB88904) and anti-αvβ3-integrin (MAB1976), goat polyclonal anti-α5β1-integrin (AB1950), rabbit polyclonal anti-α5-integrin (AB1928), rabbit polyclonal anti-α2-integrin (AB1936), and anti-α3-integrin (AB1920) were from Chemicon. Mouse monoclonal anti-human-vinculin (V9131) and rabbit polyclonal anti-Rab5 (R4654) were from Sigma-Aldrich. Rat monoclonal anti-HA (3F10) was from Roche. Rabbit polyclonal anti-GFP (A11122) and 4′,6-diamidino-2-phenylindole (DAPI) were from Molecular Probes. Goat polyclonal anti-GIPC1 (ab5951) and rabbit polyclonal anti-Myo6 (ab11096) were from Abcam. Streptavidin–horseradish peroxidase was from Amersham. Mouse monoclonal anti-active-α5-integrin, SNAKA51, was previously described .Human plasma FN was from Tebu-bio. Human plasma vitronectin, Engelbreth-Holm-Swarm murine sarcoma laminin, and calf skin collagen type I were from Sigma-Aldrich. Recombinant human VEGF-A165 was from Invitrogen. Recombinant human SEMA3A and mouse Sema3F were from R&D Systems. Sulfo-NHS-SS-Biotin was from Pierce.mNrp1 deletion constructs were generated by standard PCR protocols according to the Taq polymerase manufacturer's instructions (Fynnzymes) and using an HA-tagged version of full-length mNrp1 kindly donated by A. Püschel  as template. Cytoplasmic domains and the last three amino acids SEA were deleted using the following oligonucleotide primers: (i) 5′-cgccatggagagggggctgccgttg-3′ (Fw); (ii) 5′-ccaacaggcacagtacag-3′ (Re1) to amplify the mNrp1 deleted of the cytoplasmic domain; (iii) 5′-gtaattactctgtgggttc-3′ (Re2) to amplify the mNrp1 deleted of the three amino acids SEA. The corresponding PCR product was first thymidine–adenine (TA)-cloned into pCR2.1TOPO (Invitrogen) and subsequently subcloned in PINCO retrovirus or pAcGFP-N1 Vector (BD Bioscience) whose GFP coding sequence was previously substituted with the cDNA of mRFP, a kind gift of R. Tsien . α5-GFP and Rab5-GFP constructs were kindly provided, respectively, by A.F. Horwitz  and M. Zerial .The α5-PA-GFP construct was previously described [Hemagglutinin-tagged escribed .4 cells/well. Oligofection of siRNA duplexes was performed according to the manufacturer's protocols. Briefly, human ECs were transfected twice (at 0 and 24 h) with 200 pmol of siCONTROL nontargeting siRNA (as control), siGENOME SMART pools (in the case of hGIPC1 and hMyo6), or a mix of three (in the case of hNrp1) siRNA oligonucletides (Dharmacon). After 24 h (in the case of hNrp1) or 96 h (in the case of hGIPC1 or hMyo6) had passed since the second oligofection, ECs were lysed or tested in functional assays. In the case of hNrp1, the single oligonucleotide sequences were: (1) 5′-AAUCAGAGUUUCCAACAUA-3′; (2) 5′-GAAGGAAGGGCGUGUCUUG-3′; (3) 5′-GUGGAUGACAUUAGUAUUA-3′.The day before oligofection, ECs were seeded in six-well dishes at a concentration of 10 × 10Six-thousand ECs were resuspended in 0.1 ml of EBM-2 (Clonetics) with or without appropriate stimuli  and plated on 96-well microtiter plates (Costar) that were previously coated with ECM proteins at different concentrations and then saturated with 3% bovine serum albumin. After 15 min of incubation at 37 °C, cells were fixed in 8% glutaraldehyde and then stained with 0.1% crystal violet in 20% methanol. Cells were photographed with a QICAM Fast 1394 digital color camera (QImaging) and counted by means of Image-ProPlus 6.2 software (Media Cybernetics). In adhesion assays with NIH 3T3 fibroblasts or CHO cells, Dulbecco's modified Eagle's medium was used.−1 pepstatin; 50 mg ml−1 leupeptin; 10 mg ml−1 aprotinin; 2 mM phenylmethanesulfonylfluoride (PMSF); 2 mM MgCl2). Cells were lysed in buffer, incubated for 20 min on wet ice, and then centrifuged at 15,000g, 20 min, at 4 °C. The total protein amount was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce). Equivalent amounts  of protein were immunoprecipitated for 1 h with the antibody of interest, and immune complexes were recovered on Protein G-Sepharose . Immunoprecipitates were washed four times with lysis buffer, twice with the same buffer without Tween-20, and then separated by SDS-PAGE. Proteins were then transferred to a Hybond-C extra nitrocellulose membrane (Amersham), probed with antibodies of interest, and detected by an enhanced chemiluminescence technique (PerkinElmer).Endothelial cells were lysed in buffer containing 25 mM Tris-HCl, pH 7.6, 100 mM NaCl, 0.15% Tween-20, 5% glycerol, 0.5 mM ethylene glycol tetraacetic acid (EGTA), and protease inhibitors , and 1 mM PMSF. Lysates were cleared by centrifugation at 12,000g for 20 min. Supernatants were corrected to equivalent protein concentrations by BCA assay, and integrins were isolated by immunoprecipitation and analyzed by SDS-PAGE.Integrin traffic assays were performed as previously described by Roberts et al.  with minlater solution (Ambion), kept at 4 °C for 24 h, and frozen at −80 °C. After the cells were thawed on ice, total RNA was extracted following the manufacturer's recommended protocol . The quality and integrity of the total RNA were quantified by means of the RNA 6000 Nano Assay kit in an Agilent 2100 bioanalyzer (Agilent Technologies). cDNAs were generated from 1 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).Cells were placed in RNAmRNA expression of FN and endogenous control genes, i.e., 18S rRNA, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and TATA binding protein (TBP), was measured in the samples by real-time RT-PCR using TaqMan Gene Expression Assays run on an ABI PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems). The following assays were used: Hs00365058_m1 (FN), Hs99999901_s1 (18S rRNA), Hs99999905_m1 (GAPDH), and Hs00427620_m1 (TBP). Three replicates were run for each gene for each sample in a 384-well format plate (cDNA concentration 20 ng/well) according the manufacturer's protocol. Between the three measured endogenous control genes, we chose TBP for normalization, identified by geNorm . The expmNrp1-mRFP and Rab5-GFP were then washed in PBS, incubated with 10 μg/ml SNAKA51 Ab in EBM-2 for 30 min at 4 °C, washed 3 times in PBS, transferred to prewarmed EGM-2, and allowed to recover at 37 °C for 2 min to induce endocytosis. Cells were washed in PBS, fixed in 4% paraformaldehyde, permeabilized in 0.01% saponin for 10 min on ice, and incubated or not with the Alexa-Fluor-405-conjugated secondary antibody (Molecular Probes) for 1 h at room temperature. Cells were analyzed by using a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems). Immunofluorescence analysis was performed as previously described [Cells were plated on glass coverslips coated with 1 μg/ml FN (TebuBio) and allowed to adhere for 3 h. In addition, ECs cotransfected with escribed .4 cells/well and left to adhere for 3 h in EGM-2 medium (Clonetics) containing FN-depleted serum. Cells were then washed with PBS and fixed with 3.7% paraformaldehyde for 20 min at room temperature. Next, cells were permeabilized in PBS containing 0.1% Triton X-100 on wet ice for 2 min and incubated with anti-FN Ab for 1 h at room temperature. After three washes, cells were incubated with anti-mouse Alexa Fluor 555 for 45 min at room temperature and subsequently with DAPI. Cells were finally examined using a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems).Small interfering RNA silencing was performed, and after the second oligofection, cells were seeded onto glass coverslips in six-well dishes at a concentration of 20 × 10Fluorescence resonance energy transfer was detected using a Lambert Instruments fluorescence attachment (LIFA) on a Nikon Eclipse TE 2000-U microscope, with the same changes to the condenser as described above and a filter block consisting of a Z473/10 excitation filter, a Z 488 RDC dichroic mirror, and a HQ 525/50M emission filter. The light source was a modulated 473-nm laser diode, which allows, in combination with the modulated intensifier from the LIFA system, measurement of fluorescence lifetimes using frequency domain. The laser was brought into TIRF mode before acquiring the images for the lifetime analysis.mNrp1-Cherry transfected NIH 3T3 cells. For statistical evaluation, results were analyzed with Student's t test.Donor (D) lifetime, τ, was analyzed either in the presence or in the absence of the acceptor (A), in adhesion sites, characterized by high donor concentrations, using the FLIM software . Fluorescence resonance energy transfer efficiency (E) was calculated as E = 1 – (τDA/τD). Lifetime τ was evaluated in four different areas (12 × 12 pixels) of seven α5-GFP and seven α5-GFP/mNrp1-Cherry using 473- and 561-nm excitation. A Multi-Spec dual emission splitter  with a 595-nm dichroic and two bandpass filters (510–565 nm for green and 605–655 nm for red) was used to separate both emissions. All cell imaging was performed with a Cascade 512F EMCCD camera (Photometrics UK).Total internal reflection fluorescence experiments have been performed on a Nikon Eclipse TE 2000-U microscope equipped with 60× and 100× 1.45 NA Nikon TIRF oil immersion objectives. The Nikon Epi-fluorescence condenser was replaced with a custom condenser in which laser light was introduced into the illumination pathway directly from the optical fiber output oriented parallel to the optical axis of the microscope. The light source for evanescent wave illumination was either a 473-nm diode, a 405-nm diode, or a 561-nm laser (Omicron), with each laser line coupled into the condenser separately to allow individual TIRF angle adjustments. Each laser was controlled separately by a DAC 2000 card or a uniblitz shutter operated by MetaMorph (Molecular Devices). A filter block consisting of an E480SPX excitation filter, a FF 495 dichroic mirror, and an ET 525/50M emission filter was used for activation of α5-PA-GFP with the 405-nm laser. After activation the filter was manually changed to a green/red dual filter block  to allow simultaneous time-lapse acquisition of activated α5-PA-GFP and mNrp1-Cherry-positive vesicles was done on a FV 1000 Olympus confocal microscope, using two-channel imaging and a separate SIM scanner for 405-nm activation [Localized activation of α5-PA-GFP in tivation .Figure S1hMyo6 , but neither hNrp1  nor hGIPC1 , silencing reduce FN mRNA and protein levels. Vinculin was used as normalizer protein in Western blot analysis to calculate normalized optical density (NOD) units.(A–F) Real-time RT-PCR (A–C) and Western blot analyses (D–F) on total RNAs and proteins extracted at different times of cell spreading in the absence of exogenously added extracellular matrices reveal that hGIPC1 (H), or sihMyo6 (I) transfected ECs. DAPI was used to stain nuclei. White bar in (I) corresponds to 50 μm.(G–I) Confocal scanning microscopy analysis of endogenous FN fibrils in siCtl (G), si(10.0 MB TIF)Click here for additional data file.Figure S2mNrp1, mNrp1dSEA, or mNrp1dCy overexpression does not affect the adhesion of NIH 3T3 fibroblasts to VN.(825 KB TIF)Click here for additional data file.Figure S3hNrp1) does not alter α5 integrin expression compared with that of control silenced cells (siCtl).Immunoprecipitation of either total or active α5β1 followed by Western blot analysis for α5 shows that silencing Nrp1 in human ECs (si(1.5 MB TIF)Click here for additional data file.Figure S4As in (9.6 MB TIF)Click here for additional data file.Figure S5mNrp1-Cherry and α5-PA-GFP. α5-PA-GFP integrin fluorescence was then locally photoactivated in an mNrp1-Cherry-positive area devoid of vesicles (blue circle) and followed in time-lapse confocal microscopy. Fluorescence intensity was measured over time outside of Nrp1-positive vesicles (blue circle) and at the plasma membrane (red circle). The time-lapse plot (lower panel) shows that, under the same experimental conditions used in the experiment shown in NIH 3T3 cells were cotransfected with (9.4 MB TIF)Click here for additional data file.Video S1mNrp1-Cherry and α5-PA-GFP, photoactivated in TIRF, and observed in time-lapse epifluorescence microscopy.As in (4.3 MB MOV)Click here for additional data file.Video S2(3.1 MB MOV)Click here for additional data file.Video S3mNrp1-Cherry and α5-PA-GFP. α5-PA-GFP was locally photoactivated in vesicles containing mNrp1-Cherry and followed by time-lapse confocal microscopy.As in (9.7 MB MOV)Click here for additional data file.Video S4mNrp1-Cherry and α5-PA-GFP. α5-PA-GFP was locally photoactivated in an mNrp1-Cherry-positive area devoid of vesicles and followed by time-lapse confocal microscopy.As in (2.6 MB MOV)Click here for additional data file.


Leifsonia xyli is a xylem-inhabiting bacterial species comprised of two subspecies: L. xyli subsp. xyli (Lxx) and L. xyli subsp. cynodontis (Lxc). Lxx is the causal agent of ratoon stunting disease in sugarcane commercial fields and Lxc colonizes the xylem of several grasses causing either mild or no symptoms of disease. The completely sequenced genome of Lxx provided insights into its biology and pathogenicity. Since IS elements are largely reported as an important source of bacterial genome diversification and nothing is known about their role in chromosome architecture of L. xyli, a comparative analysis of Lxc and Lxx elements was performed.Lxc genome and comparative analysis with Lxx complete DNA sequence revealed a variable number of IS transposable elements acting upon genomic diversity. A detailed characterization of Lxc IS elements and a comparative review with IS elements of Lxx are presented. Each genome showed a unique set of elements although related to same IS families when considering features such as similarity among transposases, inverted and direct repeats, and element size. Most of the Lxc and Lxx IS families assigned were reported to maintain transposition at low levels using translation regulatory mechanisms, consistent with our in silico analysis. Some of the IS elements were found associated with rearrangements and specific regions of each genome. Differences were also found in the effect of IS elements upon insertion, although none of the elements were preferentially associated with gene disruption. A survey of transposases among genomes of Actinobacteria showed no correlation between phylogenetic relatedness and distribution of IS families. By using Southern hybridization, we suggested that diversification of Lxc isolates is also mediated by insertion sequences in probably recent events.Sample sequencing of Lxc and Lxx. The IS elements were probably acquired after the divergence of the two subspecies and are associated with genome organization and gene contents. In addition to enhancing understanding of IS element dynamics in general, these data will contribute to our ongoing comparative analyses aimed at understanding the biological differences of the Lxc and Lxx.Collectively our data indicate that transposable elements are involved in genome diversification of  Leifsonia xyli comprises two subspecies: L. xyli subsp. xyli (Lxx) and L. xyli subsp. cynodontis (Lxc). In its unique natural host, Lxx causes ratoon-stunting disease, a malady that affects sugarcane commercial fields worldwide, promoting losses of up to 30% in susceptible varieties [Lxx genome has provided important insights into the biology and pathogenicity of this bacterium [Lxc is an endophyte of Bermuda grass (Cynodo dactylon) and, when artificially inoculated, can grow in and colonize the xylem of agriculturally important grasses , causing no (or mild) symptoms of disease [Lxc may be a potential vector for expressing heterologous proteins in plants [Lxc and Lxx by sample sequencing the Lxc genome. Our goal is to comprehensively assess gene content and genomic organization of these two closely related bacteria to enhance understanding of the differences in their pathogenicity and host range. Here, we present the in silico characterization of insertion sequence (IS) elements, the most abundant type of mobile genetic element found in L. xyli [Lxc and Lxx genome diversification.The Gram-positive, coryneform, fastidious, xylem-inhabiting bacteria arieties . Sequencacterium . Lxc is  disease . Some stn plants -10,4. We L. xyli  and theiIS elements are small transposable DNA fragments ranging from 0.7 to 3.5 kbp, comprising a transposase-encoding gene and terminal inverted repeats (IR) . Close tLxx1–5) in the Lxx genome were reported, along with 47 other transposase-related genes from uncharacterized elements [Lxc, three IS elements have previously been identified: IS1237 [Lxc1 and ISLxc2 [Lxc IS elements (ISLxc3 and ISLxc4) and one new Lxx element (ISLxx6) is presented, as well as a comparative review of all elements found in both genomes, their distribution among families [Lxx and Lxc, with their influence most likely occurring after the divergence of the two subspecies from their common ancestor.Previously, 50 copies of five distinct IS elements ; IS256 family (two elements); and IS481 family (one element). Elements of the same families were not characterized in Lxx as well [Lxc genome.Among all the contigs, 70 shared sequence similarity to transposase-encoding genes. Fifty-six of these represented copies of five distinct IS elements that were characterized based on the criteria proposed Table . The remes Table : IS3 fam as well . These uLxx elements , which was not identified in our Lxc dataset.The transposases of characterized IS elements shared less than 78% amino acid sequence identity with transposases of es Table , with thLxc: IS1237 (GenBank ID: X75973); ISLxc1 (GenBank ID: EF437436); and ISLxc2 (GenBank ID: EF176596), we identified two new elements, named ISLxc3 (GenBank ID: EF421582) and ISLxc4 (GenBank ID: EF433175)  is read-through . However, they share conserved amino acids with elements of group IS1031   and the other three copies (896 bp) showed the same deletion within the core region, which were considered shorter versions of the ISLxc4 (ISLxc4d1 – GeneBank ID: EF494674). The IRs are twenty-eight bp long, and no DR was identified flanking the occurrences. Regardless of been unusual for IS30 family elements, the long version contain a cluster of purines (nucleotide position 431–437), resembling a slippery codon for the frameshift  DNA-binding domain at the N-terminal part and the well-conserved D-(54–61 aa)-D-(33 aa)-E motif at the C-terminal part [Lxc4, if the +1 frameshift does occur, the transposase (OrfAB) is 380 amino acids long and contains two domains. If the frameshift does not occur, the transposase (OrfA) is 115 amino acids long, carrying only the DNA-binding domain. Truncated transposases with only the DNA-binding domain may function as negative regulators of transposition when interacting with IRs, resulting in either repression of the transposase gene promoter or competition with full-length transposases [ISnal part . In ISLx61 aa)-D- aa-E motsposases . NeitherLxc and Lxx is 93%, as calculated on 80 kbp of 25 continuous Lxc-contigs, which were physically linked based on scaffold orientation. Comparing the adjacent regions of all 56 Lxc-IS element with the Lxx sequence, 44 elements could be mapped onto the Lxx genome , Lxx17040  and Lxx21675 ; another one, Lxx01850 , presented a conserved acyltransferase domain, associated with lipopolysaccharide modification; and the last one, Lxx14740 , presented a DNA-binding domain. Within Lxc, those genes were invaded by IS1237, ISLxc2, ISLxc3, IS1237 and ISLxc4, respectively. No DRs were detected within Lxx orthologous that could be an indicative of a prior invasion and excision event. Therefore is more likely that these genes were truncated prior to the IS elements insertion in Lxc.To assess the impact of IS elements in gene disruption, they were classified into three categories: IS insertions within predicted genes; insertions in non-coding sequences; and insertions in non-coding sequences, but with one or two truncated genes nearby (less than 100 nucleotides distant) Fig. . Truncat5 family are common in both Archaea and Eubacteria [Leifsonia xyli, examining ISfinder submission reports and using sequence similarity searches, we found that IS5 elements were the least represented in the sequenced genomes of Actinobacteria, despite being the most highly represented IS element in the Lxc genome. As observed in L. xyli genomes, transposases from the IS30 and IS481 families are abundant in most Actinobacteria  as compared to the Lxc genome (53%) is probably due to the Lxx-genome decay process, since a higher percentage of genes were already non-functional, as previously proposed [Lxc and Lxx genome comparative arrangement was assessed by mapping the flanking sequences of each Lxc-IS element in the complete Lxx sequence; some elements were associated with DNA rearrangements and others were inserted into specific fragments of the Lxc genome.Apart from sharing the same IS families, there were no iso-forms in common between the proposed . Inserti1237 is an unique element, with some similarity to elements of Streptomyces coelicolor A3(2) (NP_624431), S. avermitilis MA-4680 (NP_821293) and Frankia sp. CcI3 (YP_481128). An expansion of it was detected in the Lxc chromosome, whereas elements of the IS5 family were the least represented in Actinobacteria. Particularly intriguing is the presence of a single copy of an IS1237-related element within Lxx genome, the ISLxx6. Despite sharing 85% of nucleotide identity with IS1237, the transposase of ISLxx6 appears to be intact. One would probably expect a larger expansion of it within Lxx genome, but somehow its transposition may be controlled by mechanisms other than that regulated at translational level. This is probably an interesting model to be further analyzed. IS elements are very often plasmid-borne, and transfer events between genomes and plasmids are common [1237, first described in the cryptic plasmid pCXC100 present in some of the Lxc isolates [Lxc and Lxx-IS elements among Actinobacteria genomes showed no correlation between phylogenetic relatedness and distribution of IS families, which is probably in agreement with the hypothesis that genomes undergo repetitive extinction-reinfection cycles of different IS elements throughout bacterial evolution [IS3(2) NP_6431, S. avolution .L. xyli were characterized by maintaining transposition at low levels using mechanisms, such as regulation at the translational level [i.e., transposition occurs only when translation overcomes features of the sequences such as sites for ribosomal slippage and premature termination codons. So far, copies of Lxc and Lxx elements sequenced within each genome have proved almost identical, therefore, we have assumed that these features are part of the mechanism to regulate transposition, rather than being indicative of defective IS elements [Most of the IS families assigned in al level , i.e., telements .L. xyli IS elements carrying these types of transposases in promoting genome reorganization, the distribution of three IS elements of different families was analyzed in two strains of Lxc. The observed band polymorphism raised the hypothesis that ISLxc2 and ISLxc4 are involved in diversification, either by being active in transposition or by promoting homologous recombination. Variation was not observed for IS1237 hybridization, supporting previous experimental data where mobilization in vitro was not achieved [Lxc chromosome, the identical positioning in different isolates, and insertions within other elements suggest recent expansion of IS1237.To assess the involvement of achieved . ConversTransposon activity is regarded as an important source of bacterial diversity not only in promoting gene inactivation and genome reorganization but also in acquisition of new sequences . ConsequLxc was limited, an extensive study using the data gathered was sufficient to show that the genomic diversification of Lxc and Lxx is also and perhaps primarily associated with the presence of distinct types of IS elements. In addition to that, we have made a detailed characterization of IS elements present in each genome, which is often missing in analysis of fully sequenced genomes. The set of IS elements being unique to each genome, their specific location in combination with rearrangements and horizontal gene transfer are probably the major forces of genome evolution in Leifsonia xyli and consequently should have an impact on its biology. The same is probably true for isolates of Lxc as determined by our hybridization experiments. Our study also provided information in concert with the concept that distribution of IS elements in a given genome happens in evolutionary recent events due to cycles of expansion and extinction.The impact of IS elements on genomic organization and gene content among closely related species has previously been described ,17, and Lxc and Lxx genomes.These data will probably contribute to our ongoing comparative analyses aimed at understanding the biological differences of the Lxx CTCB07 (NC_006087) [Lxc DSMZ46306 (Taiwan); and Lxc SB . The DSMZ46306 strain was grown in a MSC New modified liquid medium [The bacterial strains used were: _006087)  (Brazil)d medium  at 28°C BamHI [pIndigoBAC-5 (Epicentre®). Both ends of each insert were sequenced using an automated sequencer . Results were analyzed by ABI sequencing analysis software, and assembled using the phred/Phrap/consed package [phred quality ≥ 20.Two DSMZ46306 genomic libraries were prepared. Genomic DNA extracted as previously described  was mechBamHI  cloned i package . All con-05. Positive matches for transposase/integrase were manually verified to determine the presence of the following determinants of a given family: element size; presence of terminal inverted repeats (IR) and conserved terminal based pairs; target site of insertion and number of bases associated to direct repeats (DR); number of ORFs; distance among amino acids of DDE; as well as comparisons with related elements [Inserts containing end-sequences similar to transposases were subcloned or primer-walked. Homology searches were performed using BlastN and BlastX  at GenBaelements . We alsoTransposases sharing more than 95% of amino acid identity were grouped. Their nucleotide sequences in addition to 300 bases up and downstream were aligned using ClustalW . For eacLxc-IS element loci were compared to those of the Lxx genome using adjacent sequences as anchors and a cross_match program, which is part of the swat/cross_match/phrap package [Lxc-specific loci were annotated using SABIA [ package , with deng SABIA .Amino acid sequences of putative transposases were used as queries within completely sequenced genomes of Actinobacteria. Genomes were downloaded from GenBank  was digested completely with PstI, EcoRI, BamHI and EcoRV. Fragments were separated in a 0.8% (w/v) agarose gel and transferred to a Hybond N+ membrane . Probe labeling, hybridization and detection were performed with an ECL kit (Amersham) according to the manufacturer's guidelines and under high stringency conditions. Inserts of shotgun clones containing the entire fragments of three Lxc-IS elements  were amplified and used as probes. The same membrane was stripped and re-used according to the manufacturer's recommendations.in silico analysis and drafted the manuscript. M–AVS: Data interpretation and analysis and helped to draft the manuscript. LEAC: Data interpretation and analysis and helped to draft the manuscript. CBM–V: conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.MMZ: carried out the molecular genetic studies, prepared the Lxc-IS elements and the most related IS element of Lxx genome.Comparative analysis of Click here for fileLeifsonia xyli subsp. cynodontis.Characterization of IS elements of Click here for fileLxx (gray lines) and Lxc (white lines) IS elements within the Lxx genome based on their flanking sequences.Mapping of Click here for fileLxc genome.IS elements inserted in specific fragments of Click here for fileLxx6 and IS1237 sequence alignment and putative transposase amino acid sequences.Details of ISClick here for file


P = 0.01) and stage of disease (P = 0.03) but not with serum PSA levels. In addition moderate or high RAC 3 expression was associated with poorer disease-specific survival (P = 0.03). We conclude that RAC 3 is an important co-activator of the AR in the prostate and may have an important role in the progression of prostate cancer. © 2001 Cancer Research Campaign http://www.bjcancer.comRAC 3, one of the p160 family of co-activators is known to enhance the transcriptional activity of a number of steroid receptors. As co-activators are also known to enhance androgen receptor (AR) activity, we investigated the role of RAC 3 in the context of prostate cancer. In prostate cancer cell lines, we found variable levels of the RAC 3 protein with highest expression seen in AR-positive LNCaP cells, moderate expression in AR-negative PC 3 cells and low-level expression in AR-negative DU 145 cells. Immuno-precipitation studies showed that endogenous RAC 3 interacted with the AR in vivo and transfection assays confirmed that RAC 3 enhanced AR transcriptional activity. In clinical prostate tissue, we found strong RAC 3 mRNA expression and immuno-histochemistry demonstrated that in benign tissue, the protein was expressed predominantly in luminal cells, while in primary malignant epithelium it was more homogeneously expressed. In a series of 37 patients, the levels of RAC 3 expression correlated significantly with tumour grade (