text:
BARCELONA ISSUE 14 Feb 2004
Casting La Ponderosa Barcelona models, actors, casting
Casting La Ponderosa is using a new system: DVD.
Since January 2004, La Ponderosa offers -as well as VHS, UMATIC and MiniDV- the possibility to record online castings on DVD.
It gives a lot more possibilities to production companies: The casting can be seen online from anywhere in the world. It is cheaper and more practical than UMATIC, and it gives you better quality at the same time.
La Ponderosa is also editing DVD with a specific menu for every character from different agencies, so they can show new material to their clients.
Tag your comment using @productionparadise to share with us!


text:
The recommendations you provide during the college application or scholarship application process are just as important as the application themselves. Having a list of individuals who could provide a recommendation is key to obtaining the best recommendations for your purposes. Our academic advisors are able to provide recommendations to students who have been enrolled with us for at least one full school year and are in good standing without academic warnings. If you fall into this category and would like to request a recommendation from your advisor, please take the time to fill out this form. Be as specific as possible as it will help your advisor to provide the best recommendation possible.
Recommendation Request Print
Modified on: Tue, 19 Mar, 2019 at 1:00 PM
Did you find it helpful? Yes NoSend feedback
Sorry we couldn't be helpful. Help us improve this article with your feedback.

subset: commoncrawl


text:
People of all ages, in all walks of life are interested in nutrition. Although, nutrition is still a science that needs to be explored much further. Studies are being done every day and every year to unravel all the pieces that make up the nutrition puzzle. The results will be wonderful.
Try to purchase mostly organic foods if you are eating for good nutrition. Fresh foods that have not been processed offer the best nutritional content and contain fewer chemicals and fats.
Gradually change your diet if you want to be successful with it. You can't make changes happen right away. Do not shock your system too much by consuming foods that you hate. Slowly putting healthier items into your diet over some time will help you become healthier.
Organic food is the best type of food that you can put in your body. There are copious amounts of research showing that organic foods tend to be lower in nitrates and richer in nutrients. This is what Mother Nature intended. You will have a better understanding if you give it a try.
Always bring your own meals when heading out of the house, whether for work or school. If you bring your own meal and snacks, you will be less tempted by fast food and other unhealthy options. In just a few moments you can pack and freeze lunches for an entire week.
To maximize the nutritional content of the foods in your diet, reduce the number of microwavable dinners that you eat. The reason is that such items often contain excessive quantities of sugar and fat. Purchase fresh produce and meat and cook them yourself to extract the greatest health benefits.
Your diet needs to consist of the right nutrition to enable proper body function and better health. To ensure your body is receiving the proper vitamins, take a multivitamin supplement. Any vitamin shop will be able to help you find what you need. For instance, if you're 50 years old and a female, you may need a vitamin made for females that are middle age. Drink lots of water when you take your vitamins.
In summation, nutrition changes all the time as the studies and information on it increase. The more information you have about nutrition, the better our health. Always pay attention to the latest news about nutrition.


text: Julia Bauscher Named 2014-2015 President of the School Nutrition Association FOR IMMEDIATE RELEASE: Contact: Diane Pratt-Heavner 703-576-7526 firstname.lastname@example.com Julia Bauscher Named 2014-2015 President of the School Nutrition Association 2014-07-16 Follow us @schoollunch and #ANC14Boston, MA (July 16, 2014) - During its 68th Annual National Conference, the School Nutrition Association (SNA) installed Julia Bauscher, SNS, as its 67th president for the 2014-2015 school year. Bauscher, the director of school and community nutrition services for Jefferson County Public School (JCPS) system in Louisville, Kentucky, takes the helm of an organization that represents 55,000 school nutrition professionals nationwide.Bauscher began a successful career in school foodservice after graduating from the University of Kentucky with a degree in dietetics. After 12 years in foodservice sales, Bauscher joined JCPS as coordinator of food procurement and has been with the district for 20 years. She held her first position with the district for five years before becoming the operations manager of the Nutrition Service Center, and took over as director of school and community nutrition services for the 145-site district in 2009. Under Bauscher's leadership, JCPS has implemented farm to school, Breakfast in the Classroom, and an at-risk supper program. The district also offers a mobile foodservice program, the Bus Stop Café, which added an additional bus to the fleet for Summer 2014. This year, JCPS is launching the Community Eligibility Provision, allowing nearly 55,000 students at 95 schools to received free breakfast and lunch, regardless of income. This not only saves families money —$122 per child a year for those that previously qualified for reduced-price meals, and $700 per child per year for those who did not—but it also removes the stigma often associated with receiving free school meals. Bauscher also has a storied history with SNA, having served at the state level as president of Kentucky SNA. On the national level, she sat on the Nutrition Committee for a number of years, including serving as its chair. She was elected Vice President for the national organization for the 2012-2013 school year and served as President-Elect in 2013-2014. About the School Nutrition Association:The School Nutrition Association (SNA) is a national, non-profit professional organization representing 55,000 school nutrition professionals across the country. Founded in 1946, SNA and its members are dedicated to making healthy school meals and nutrition education available to all students. For more information about ANC, visit www.schoolnutrition.org/anc.


text:
AK Steel Launches Innovation Hub in Middletown
- By
- Jun 22, 2015
By Adrian Maties, Associate Editor
AK Steel broke ground June 19 on its $36 million Research and Innovation Center in Middletown. James Wainscott, AK Steel's chairman, president & CEO, attended the ceremony, along with Mayor Larry Mulligan and other officials.
AK Steel announced plans for the facility earlier this year. The project calls for the construction of a 135,000-square-foot facility on 16 acres in Middletown's Renaissance District. The site is located along Interstate 75 in the Cincinnati–Dayton growth corridor.
AK Steel's Research and Innovation Center will replace an existing facility in Middletown. It will house pilot lines that simulate manufacturing operations, ranging from melting and casting and hot- and cold-rolling to final finishing. Seventy-six of the company's researchers, scientists and engineers will work in the facility. AK Steel said that it expects the project to add 15 new full-time research positions.
Al Neyer L.L.C. is building the Research and Innovation Center. Neyer Architects Inc. is the project's architect, with BHDP Architecture in charge of designing the interior labs and office areas. AK Steel expects completion by late 2016.
Photo credit: AK Steel


text:
- nounOutpatient is used before these nouns: ↑appointment, ↑care, ↑clinic, ↑department, ↑programme, ↑surgery, ↑treatmentOutpatient is used after these nouns: ↑hospital
Collocations dictionary. 2013.
Look at other dictionaries:
outpatient — (n.) 1715, person who is treated at a hospital but not admitted, from OUT (Cf. out) + PATIENT (Cf. patient) (n.). The adjective is first recorded 1879 … Etymology dictionary
outpatient — ► NOUN ▪ a patient attending a hospital for treatment without staying overnight … English terms dictionary
outpatient — [out′pā΄shənt] n. a patient who is treated at a hospital, clinic, etc. and leaves without becoming an inpatient … English World dictionary
outpatient — [[t]a͟ʊtpeɪʃənt[/t]] outpatients also out patient N COUNT: oft N n An outpatient is someone who receives treatment at a hospital but does not spend the night there. ...the outpatient clinic... She received psychiatric care as an outpatient. Ant:… … English dictionary
outpatient — UK [ˈaʊtˌpeɪʃ(ə)nt] / US noun [countable] Word forms outpatient : singular outpatient plural outpatients someone who receives medical treatment at a hospital, but who does not stay there for the night an outpatient clinic/department See:… … English dictionary
outpatient — out|pa|tient [ˈautˌpeıʃənt] n someone who goes to a hospital for treatment but does not stay for the night →↑inpatient ▪ an outpatient clinic ▪ a routine examination in outpatients (=the outpatient department of a hospital) … Dictionary of contemporary English
Outpatient — A patient who is not an inpatient (not hospitalized), but instead comes to a doctor's office, clinic, or day surgery for treatment). Outpatient care is called ambulatory care. * * * A patient treated in a hospital dispensary or clinic instead of… … Medical dictionary
outpatient — out|pa|tient [ aut,peıʃnt ] noun count someone who receives medical treatment at a hospital, but does not stay there for the night: an outpatient clinic/department ─ compare INPATIENT … Usage of the words and phrases in modern English
outpatient — noun Usage: often attributive Date: 1715 a patient who is not hospitalized overnight but who visits a hospital, clinic, or associated facility for diagnosis or treatment compare inpatient … New Collegiate Dictionary
outpatient — /owt pay sheuhnt/, n. a patient who receives treatment at a hospital, as in an emergency room or clinic, but is not hospitalized. Also, out patient. [1705 15; OUT + PATIENT] * * * … Universalium


text:
March 11, 2008
Whole Terrain on Wikipedia
Whole Terrain has been added to Wikipedia! The entry includes our mission, and lists back issues and some of our contributors. Wikipedia is open for anyone to contribute or amend. Please lend your editorial genius to any articles that connect to the Whole Terrain Wikipedia entry.


text: The Joint Commission The Joint Commission continuously improves health care for the public, in collaboration with other stakeholders, by evaluating healthcare organizations and inspiring them to excel in providing safe and effective care of the highest quality and value. Frequently Asked Questions About The Joint Commission What is The Joint Commission? An independent, not-for-profit organization, The Joint Commission is the nation's oldest and largest standards-setting and accrediting body in health care. The Joint Commission accredits and certifies more than 20,500 healthcare organizations and programs in the United States. Learn more about The Joint Commission What Is an Accreditation Survey? Joint Commission surveyors visit accredited healthcare organizations a minimum of once every 39 months (two years for laboratories) to evaluate standards compliance. This visit is called a survey. Joint Commission surveyors are highly trained experts who are: Doctors Nurses Hospital administrators Laboratory medical technologists Other healthcare professionals How Long Does It Take The Joint Commission to Render an Accreditation Decision? The Joint Commission renders accreditation decisions two weeks to two months after the survey. Where Can I Find Information About an Accredited Healthcare Organization? Joint Commission Quality Reports give the public information on the safety and quality of care for all Joint Commission accredited/certified health care organizations. Quality Reports are available online through Quality Check®. Quality Reports include: Accreditation decision and date Programs and services accredited by The Joint Commission and other bodies National Patient Safety Goal performance Hospital National Quality Improvement Goal performance Special quality awards What are the Benefits of Accreditation for Patients? The benefits include increased confidence in the care provided because you know the organization is: Continually working to provide the highest quality services Periodically evaluated by The Joint Commission for compliance with the highest quality standards Provided with expert advice and education from The Joint Commission about quality improvement Able to attract qualified staff because they have more opportunities to develop their skills and knowledge


text:
As the Swiss National Bank (SNB) gets ready to meet on Wednesday, a number of analysts say the central bank has few choices to try and stem the franc's recent rise.
"(They) can't stave off the flood of demand for Swiss francs if markets continue to deteriorate," Brian Dolan, Chief Currency Strategist at FOREX.com told CNBC on Wednesday ahead of the meeting.
The currency has surged some 20 percent against the U.S. dollar in the last 6 months and around 13 percent against the euro. Those sharp gains have hurt Swiss exporters and threatened to tip the country into recession. It's also increased speculation the SNB may peg it's currency to the euro, given that interest rates are already near zero.
"That is my least preferred option for them to take," Dolan said, arguing once the currency has been pegged, there would be no looking back. "That obligates them to use interest rates or direct market intervention to maintain whatever peg they decide to set."
BNP Paribas also thinks such direct intervention would fail to address the root cause of the franc's gains - a surge in Swiss bank deposits during global recessions. BNP points out the recent upswing in the currency started in late 2008, immediately after the collapse of Lehman Brothers. A similar trend occured between 1990 and 2001.
"The increase from 1990 led to a particularly sharp CHF appreciation. Interestingly, all three cases of surges in Swiss savings deposits have occurred during U.S. recessions," BNP said in a report.
According to FOREX.com's Dolan, a better way to tackle the problem would be to directly target these deposits with a punitive tax.
"A more likely option they're likely to pursue will be raising a tax on Swiss deposits, basically calling for negative interest rates. And that will make it a little less attractive to hold Swiss francs," he said.
BNP Paribas points out that if history is any guide, this too may not work. Between 1972 and 1978, Switzerland imposed taxes on non-resident deposits equivalent to an effective tax of 40 percent per annum but the Swiss franc soared 78 percent against the euro during that period.
"The conclusion is that the SNB will likely struggle to reverse the current bout of Swiss franc strength given the market preference for the Swiss franc as a safe haven from economic and market conditions in both the U.S. and the eurozone," BNP Paribas said.
Dolan agrees that trend will persist as long as global uncertainties remain.
"The best we can hope for, the best they can hope for, is some stabilization in global risk assets. And that will lessen the demand for Swiss franc on its own," he said.


text:
|The Five Star Management Call Sheet
|Taking Through The Call Sheet
|Financial Work Sheet Tutorial
|Spinal Screening Workshop in London
|Informal Spinal Screening Workshop in London
|Use of checklists for lead CA and COA. This was a Galaxy project. (Private)
|Office meeting with CA and two DC. (Private)
|Office Meeting #1 (Private for right now, but clients will have access in future.)
|Office meeting #2 (Private)
|Office meeting #3 (Private)
|Accounts manager meeting with AR (Private)
|Chiropractic Care Schedule (Private)
|Financial Worksheet Example (Private)
|How to Schedule Appointments (Private)


text:
Fan or fans may refer to:
One of several types of fan-shaped deposits of sediment caused by the flow of streams or glacial melt:
Fanø is a Danish island in the North Sea off the coast of southwestern Denmark, and is the very northernmost of the Danish Wadden Sea Islands. Fanø municipality is the municipality (Danish, kommune) that covers the island and its seat is the town of Nordby.
Fanø is separated from the mainland by the Wadden Sea over a span of approximately five kilometres. The island is 16 km long and 5 km wide, and it is located off the coast from the city of Esbjerg to which it is connected by ferry. The ferry ride takes 12 minutes. The high ferry ticket prices that are often subject to price increases are considered by the locals to be a great threat to the local tourism industry.
A variety of environments are to be found on Fanø. Not surprisingly, a very common one is sand. The island's whole western shore is one long beach. The island's northwestern corner is a vast sandbank called "Søren Jessens Sand". Søren Jessen was an entrepreneur and captain from Hjerting, today the westernmost suburb of Esbjerg and the bank is named after him because his ship, the "Anne Catriane", stranded here in 1712.
La Belle Lucie, The Fan, Clover Leaves, Three Shuffles and a Draw, Alexander the Great, Trefoil or Midnight Oil is a solitaire where the object is to build the cards into the foundations.
All cards are visible from the start, but this does not imply that this game is solvable with strategy. The default rule is very hard to win. The majority of games cannot be solved. For example, moving a single card onto another blocks that stack until both cards can be removed to the foundations. Any setup that has a lower card of a specific suit below a higher of the same suit, or all kings not on the bottom of each cascade cannot be solved without cheating. The shuffle and redeal is of little help. For each king left in the second redeal, there is a 66% chance that the cascade cannot be solved (if the king is not lowest). Moving aces out (Trefoil rule) has cosmetic character.
Marino, Mariño or Maryino may refer to:
As a name:
Sports teams:
Marino is a surname and a given name of Latin origin.
Marino (Marinum or Castrimoenium in Latin, Marini in local dialect) is an Italian city and comune in Lazio (central Italy), on the Alban Hills, Italy, 21 kilometres (13 miles) southeast of Rome, with a population of 37,684 and a territory of 26.10 square kilometres (10.08 sq mi). It is bounded by other communes, Castel Gandolfo, Albano Laziale, Rocca di Papa, Grottaferrata, and Ciampino. Marino is famous for its white wine, and for its Grape Festival, which has been celebrated since 1924.
The territory of Marino was inhabited by Latin tribes from the 1st millennium BC. The ancient cities of Bovillae (Frattocchie), Mugilla (Santa Maria delle Mole, a frazione of the comune of Marino) and Ferentum (Marino itself) were part of the Latin League. Under the Roman Republic it was a summer resort for Roman patricians, who built luxurious villas in the area to escape the heat of Rome.
In 846 AD, Bovillae - until then the largest settlement - was destroyed by the Saracens, and the population moved to the more easily defendable area of Ferentum, which was fortified under the new name of Marinum. From 1090 it was a dominium of the Counts of Tusculum, and later a fief of the Frangipane and, beginning in 1266, the Orsini. In 1272, San Bonaventura founded the first confraternity of Italy there.
Ele is Finnish producer Sasu Ripatti's debut full-length album as Vladislav Delay. It was released in 1999 on the Australian label Sigma Editions.
The tracks on Ele average over twenty minutes in length. Two of the tracks, "Kohde" and the title track, were reworked in shorter versions on Delay's second album Entain.
Allmusic gave it three stars, comparing his music to that of Steve Reich, Oval, Thomas Brinkmann, and Pole and calling it Delay's "defining point-of-departure in defining a new hybrid dub, minimalism, IDM, post-techno music."


text:
Mammary epithelial cells undergo changes in growth, invasion, and differentiation throughout much of adulthood, and most strikingly during pregnancy, lactation, and involution. Although the pathways of milk protein expression are being elucidated, little is known, at a molecular level, about control of mammary epithelial cell phenotypes during normal tissue morphogenesis and evolution of aggressive breast cancer. We developed a murine mammary epithelial cell line, SCp2, that arrests growth and functionally differentiates in response to a basement membrane and lactogenic hormones. In these cells, expression of Id-1, an inhibitor of basic helix-loop-helix transcription factors, declines prior to differentiation, and constitutive Id-1 expression blocks differentiation. Here, we show that SCp2 cells that constitutively express Id-1 slowly invade the basement membrane but remain anchorage dependent for growth and do not form tumors in nude mice. Cells expressing Id-1 secreted a ∼120-kDa gelatinase. From inhibitor studies, this gelatinase appeared to be a metalloproteinase, and it was the only metalloproteinase detectable in conditioned medium from these cells. A nontoxic inhibitor diminished the activity of this metalloproteinase in vitro and repressed the invasive phenotype of Id-1-expressing cells in culture. The implications of these findings for normal mammary-gland development and human breast cancer were investigated. A gelatinase of ∼120 kDa was expressed by the mammary gland during involution, a time when Id-1 expression is high and there is extensive tissue remodeling. Moreover, high levels of Id-1 expression and the activity of a ∼120-kDa gelatinase correlated with a less-differentiated and more-aggressive phenotype in human breast cancer cells. We suggest that Id-1 controls invasion by normal and neoplastic mammary epithelial cells, primarily through induction of a ∼120-kDa gelatinase. This Id-1-regulated invasive phenotype could contribute to involution of the mammary gland and possibly to the development of invasive breast cancer.
The epithelial cells of the mammary gland undergo coordinate changes in growth, differentiation, and invasion of the surrounding ECM during embryonic development and puberty, and throughout much of adulthood during each menstrual cycle. Particularly striking changes occur during pregnancy, lactation, and involution. The molecular mechanisms that control the growth and functional differentiation of mammary epithelial cells are slowly being elucidated, but far less is known about the transient invasive behavior of normal breast epithelial cells.
Normal breast epithelial cells proliferate and invade the surrounding ECM during the fetal and postnatal development of the gland, and then more vigorously at puberty as the branches of the mammary epithelial tree are formed. After puberty, there are minor waves of mammary epithelial-cell proliferation during each estrous cycle (16, 46). The most striking activity of mammary epithelial-cell proliferation and invasion occurs during pregnancy, as the gland expands in preparation for lactation (45). The proliferation and invasion of breast epithelial cells cease during late pregnancy, whereupon the cells functionally differentiate—that is, they express and secrete milk proteins (44). The epithelial cells remain proliferatively quiescent and functionally differentiated throughout lactation. At the end of lactation, the mammary gland undergoes involution, during which time there is an early and transient reactivation of epithelial-cell proliferation, followed by extensive ECM degradation and epithelial-cell death by apoptosis. The extensive remodeling of the mammary gland that occurs during involution entails the stepwise activation of several MMPs by the stromal and epithelial cells of the gland (29, 41). The involuting gland eventually returns to its prepregnancy structure.
Invasion of the ECM by normal epithelial cells must be tightly regulated and self-limiting. This control is clearly important for the mammary gland to develop and function normally. Control over normal invasive properties is also important in order to prevent neoplastic cells from invading the surrounding ECM. Most cancers develop from epithelial cells, and a hallmark of malignancy is invasion of the ECM by neoplastic epithelial cells (38). In many experimental models of tumorigenesis, an invasive phenotype develops subsequent to neoplasia and often entails expression of ECM-degrading enzymes commonly expressed by mesenchymal or stromal cells. These enzymes include the MMPs stromelysin and the 72- and 92-kDa collagenases (19, 48). It is not clear whether tumor cells express these MMPs because they are normally expressed when epithelial cells transiently invade the ECM during normal tissue morphogenesis or because they frequently acquire mesenchymal characteristics upon transformation. It was recently shown by in situ hybridization that these MMPs are expressed by stromal fibroblasts during certain stages of ductal and alveolar mammary morphogenesis as well as during involution (29, 49).
In order to study normal and abnormal mammary epithelial-cell phenotypes, we developed a murine mammary epithelial-cell line, SCp2, whose growth and differentiation can be controlled in culture (8). SCp2 cells are an immortal line that originated from a heterogeneous cell population derived from a midpregnancy mouse mammary gland (7, 37). SCp2 cells grow well in serum on tissue culture plastic, where they express keratins and exhibit other epithelial characteristics. When serum is removed and they are given lactogenic hormones (insulin, prolactin, and hydrocortisone) and basement membrane components, SCp2 cells first arrest growth, then aggregate and form alveolar structures, and finally express high levels of several milk proteins (8, 36).
We have shown that the differentiation of SCp2 cells requires a sharp decline in the expression of the HLH protein Id-1 (9). Id genes encode a small family of proteins that prevent bHLH transcription factors from binding DNA (4). bHLH transcription factors comprise a large family of sequence-specific DNA binding proteins that activate the transcription of cell- and tissue-specific genes. bHLH proteins act as obligate dimers: they dimerize through the HLH domains and bind DNA through the composite basic domain. Id proteins contain HLH domains and therefore dimerize with bHLH proteins. However, because Id proteins lack basic domains, Id-bHLH heterodimers cannot bind DNA. Thus, Id proteins negatively regulate bHLH transcription factors. The bHLH superfamily contains both ubiquitous and lineage-specific transcription factors that direct many developmental and differentiation processes (20). Two of the four known Id proteins (Id-1 and Id-3) are nearly ubiquitously expressed, whereas the other two Id proteins (Id-2 and Id-4) have a more restricted pattern of expression (35). Thus, lineage-specific differentiation is determined by tissue-specific bHLH genes, which, in turn, are posttranslationally regulated by a small number of Id genes. Whether and how bHLH proteins participate in the differentiation of breast epithelial cells is not yet known.
Id-1 was the first Id protein to be identified (4). Since its initial discovery in myoblasts, it has been shown to be expressed by a variety of cell types and to inhibit the differentiation of myoblasts (18), several hematopoietic cell types (23, 26, 40), trophoblasts (6), and mammary epithelial cells (9). Id-1 was also found to be serum inducible in fibroblasts, where its expression is essential for progression into the S phase of the cell cycle (14). In contrast to the closely related Id-2 protein, Id-1 does not physically associate with the retinoblastoma tumor suppressor protein pRb (15, 17) but can functionally interact with a pRb-regulated pathway for entry into S phase (15).
Id-1 expression declines rapidly when SCp2 cells are induced to differentiate. As long as the cells remain in contact with a basement membrane and lactogenic hormones, Id-1 remains repressed and the cells do not proliferate, but they express milk proteins. By contrast, SCp2 cells that constitutively express Id-1 fail to differentiate, as judged by the expression of milk proteins, but nonetheless transiently arrest growth and form loose alveolar structures. After several days, cells that constitutively express Id-1 dissociate from each other and subsequently resume growth (9).
Here, we show that Id-1 expression confers upon SCp2 cells the ability to migrate and invade the basement membrane. However, cells that constitutively express Id-1 neither grow in soft agar nor form tumors in nude mice. Id-1 expression correlates strongly with expression of an apparently novel gelatinase of approximately 120 kDa, an MMP, which is also expressed during involution. The activity of this MMP was critical for the Id-1-regulated invasive phenotype. We also show that Id-1 expression correlates with the degree of differentiation and invasiveness of human breast cancer cells. The least-differentiated and most highly invasive cells express constitutively high levels of Id-1 and also secrete a 120-kDa gelatinase. Our results suggest that Id-1 is a regulator of the invasive phenotype of normal and neoplastic mammary epithelial cells and that it acts, at least in part, by controlling expression of a 120-kDa gelatinase. The invasive phenotype conferred by Id-1 is not a consequence of tumorigenic transformation, although it may be appropriated in a subset of aggressive breast cancers. Our data provide new insights into the control of breast epithelial-cell invasion and suggest that one or more bHLH transcription factors may repress the invasive phenotype in normal as well as neoplastic breast epithelial cells.
Abbreviations.AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride; bHLH, basic helix-loop-helix; BSA, bovine serum albumin; DAPI, 4′,6′-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; EHS, Englebreth Holm Swarm tumor; ECM, extracellular matrix; F12, Ham's F-12 medium; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HLH, helix-loop-helix; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; RT-PCR, reverse transcription-PCR.
Cell culture.SCp2 cells were grown in a 1:1 mixture of DMEM and F12 (DMEM-F12) containing 5% heat-inactivated FBS, insulin (5 μg/ml), and gentamicin (50 μg/ml) (growth medium) at 37°C in a humidified 5% CO2 atmosphere, as previously described (8). To induce differentiation, cells were plated at 5 × 104/cm2 atop basement membrane components in DMEM-F12 lacking serum but containing lactogenic hormones (insulin, 5 μg/ml; hydrocortisone, 1 μg/ml; prolactin, 3 μg/ml) (9). Unless otherwise indicated, cells were cultured for 5 days before analysis. Basement membrane ECM either was purified from EHS tumors by the method of Taub et al. (42) or was supplied as Matrigel from Collaborative Research.
SCp2 cells were transfected with the murine Id-1 cDNA driven by the mouse mammary tumor virus promoter as previously described (9). The transfected cells were initially pooled. Single cell-derived clones were subsequently derived by plating cells at limiting dilutions in 24-well plates. After 10 days, wells with visible colonies were trypsinized and replated onto 35-mm-diameter dishes. When nearly confluent, the cells were replated onto 100-mm-diameter dishes. The population was expanded by subculturing at a ratio of 1:4, and cells were used after 5 to 8 passages after the first 1:4 subculture.
The human breast cancer cell lines T47D, MCF-7, Hs578T, BT-549, MDA-MB-231, ZR75-1, and SKBR-3 were purchased from the American Type Culture Collection. The MDA-MB-436 cell line was originally purchased from the American Type Culture Collection and was given to us by R. Lupu (Berkeley National Laboratory). MDA-MB-435 cells were derived from the original cell line by selection in nude mice for the highly aggressive subpopulation (37a). Cells were passaged in DMEM containing 10% FBS and insulin (5 μg/ml; Sigma). For serum-free conditions, FBS was omitted from the medium.
DNA synthesis and autoradiography.Cells plated on coverslips were labeled with [3H]methylthymidine (10 μCi/ml; 60 to 70 Ci/mmol) for 24 h, washed twice with PBS, then fixed for 5 min with a 1:1 (vol/vol) mixture of acetone and methanol at −20°C. Where indicated, cell nuclei were stained for 2 min with DAPI diluted 1:10,000 in PBS. The coverslips were air dried, coated with Kodak NTB2 emulsion (1:2 dilution), and exposed for 16 to 24 h. The coverslips were developed with D-19, fixed with Kodak Rapid-Fix, and viewed by phase-contrast microscopy.
Boyden chamber invasion assays.Invasion assays were performed in modified Boyden chambers with 8-μm-pore-size filter inserts for 24-well plates (Collaborative Research). Filters were coated with 10 to 12 μl of ice-cold basement membrane ECM at 8 to 12 mg of protein/ml. Cells (0.5 × 105 to 1 × 105) were added to the upper chamber in 200 μl of DMEM-F12. The lower chamber was filled with 300 μl of NIH 3T3 cell-conditioned medium. Where indicated, GM6001 was added at 0.2 mM to both chambers immediately after cell plating. After a 16- to 20-h incubation, the cells were fixed with 2.5% glutaraldehyde in PBS and stained with 0.5% toluidine blue in 2% Na2CO3. Cells that remained in the basement membrane or attached to the upper side of the filter were removed with paper towels. Cells on the lower side of the filter were examined by light microscopy and counted.
Anchorage-dependent growth assays.Liquefied 2% agarose was mixed with an equal volume of 2× DMEM-F12 growth medium lacking serum and supplemented with insulin (10 μg/ml) and gentamicin (100 μg/ml) (2× medium). One milliliter of the mixture was layered onto 35-mm-diameter dishes to create a 1% agarose base. Liquefied 0.6% agarose was mixed with an equal volume of 2× medium, and 10 ml of this solution was mixed with 1 ml of growth medium containing 105 cells to yield 104 cells/ml in 0.27% agarose; 1 ml of this cell suspension was layered on top of the 1% agarose base, and 1 ml of DMEM-F12 containing 5% FBS was added. The cells were incubated for 14 days, after which representative fields were photographed under phase-contrast microscopy.
Tumorigenicity assays.Cells were injected subcutaneously into nude mice at 4 × 106 cells per site, two sites per animal, and two animals for each cell type (TCL1, SCg6, SCp2, SCp2–antisense Id-1, and SCp2–Id-1). Animals injected with TCL1 and SCg6 cells developed easily detectable tumors (at least 1 cm3) within 3 weeks and were sacrificed after 4 weeks. The remaining animals remained tumor negative for a minimum of 5 months.
Immunofluorescence.Cells cultured on coverslips were washed with PBS, fixed for 5 min with acetone-methanol (1:1, vol/vol) at −20°C, permeabilized for 5 min with 1% Triton X-100 in PBS, and washed with PBS. A rabbit polyclonal antiserum raised against bovine keratins (Dako, Carpinteria, Calif.) was diluted 1:10 in 0.2% BSA in PBS and applied for 60 min at 37°C, followed by three washes in PBS. The coverslips were then incubated with biotin-conjugated anti-rabbit antibody (1:100 dilution; Amersham Corp.) for 30 min at 37°C and were washed three times in PBS. Finally, the coverslips were incubated with fluorescein isothiocyanate-conjugated streptavidin (1:100 dilution; Amersham Corp.) for 30 min at 37°C and were washed in PBS. Cell nuclei were stained with DAPI, as described above, and the coverslips were mounted in glycerol-gelatin (Sigma) for viewing by epifluorescence.
RNA isolation and analysis.Total cellular RNA was isolated and purified as described by Chomczynski and Sacchi (5). The RNA (10 μg) was size fractionated by electrophoresis through formaldehyde-agarose gels and transferred to a nylon membrane (Hybond N from Amersham Corp.). The membrane was hybridized to32P-labeled probes prepared by random oligonucleotide priming, washed, and exposed to XAR-5 film for autoradiography as described by Maniatis et al. (31). The β-casein probe was the 540-bp mouse cDNA (from J. Rosen, Baylor College of Medicine, Houston, Tex.), and the Id-1 probe was either the murine Id-1 cDNA (4) or the human Id-1 cDNA (14).
RT-PCR and Southern analysis.Transcripts for murine gelatinases A (72-kDa MMP) and B (92-kDa MMP) were detected by RT-PCR. cDNA was synthesized from total RNA by using SuperscriptII Reverse TranscriptaseII (Gibco-BRL), and 100 ng was used for PCR. The 5′ and 3′ PCR primers were TTGAGAAGGATGGCAAGTATGG and ACACCTTGCCATCGTTGC for gelatinase A, GGCGTGTCTGGAGATTCGA and AGGGTCCACCTTGGTCACC for gelatinase B, and ACCACAGTCCATGCCATCAC and TCCACCACCCTGTTGCTGTA for GAPDH. PCR was performed in 20 mM Tris-HCl (pH 8.8)–2 mM MgSO4–10 mM KCl–10 mM (NH4)2SO4–0.1% Triton X-100–100 μg of BSA/ml–0.125 mM deoxynucleoside triphosphates–0.8 μM each PCR primer–0.05 U of Pfu DNA polymerase/μl by using 35 cycles for amplification of gelatinase cDNAs and 25 cycles for amplification of GAPDH cDNA. The cycle conditions were 1 min of denaturation at 94°C, 1 min of annealing at 58°C, and 30 s of extension at 72°C. For Southern analysis, one-fifth of the PCR reaction product was separated on a 2.1% agarose gel, transferred to a nylon membrane (Hybond N+), and hybridized with cDNA inserts labeled with 32P by random priming. cDNAs encoding murine gelatinase A or B (33, 34) were a gift from Z. Werb, University of California, San Francisco, and the GAPDH cDNA was obtained from Clontech (Palo Alto, Calif.). Hybridization was carried out in 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5× Denhardt's solution, 0.5% sodium dodecyl sulfate, and 50% formamide at 42°C overnight. The membranes were washed at a final stringency of 0.2× SSC and 0.1% sodium dodecyl sulfate at 68°C and were exposed to XAR-5 film for autoradiography.
Zymography.Proliferating cells (106 in 100-mm-diameter dishes) were shifted to serum-free medium for 2 to 3 days, after which they were given 10 ml of fresh serum-free medium. Forty-eight hours later, the conditioned medium was collected and concentrated 10- to 15-fold by using 10-kDa-cutoff filters (Millipore, Bedford, Mass.). The concentrated medium was analyzed on casein and gelatin substrate gels, as described by Fisher and Werb (10) and Talhouk et al. (41). Briefly, gels consisted of 8 to 10% polyacrylamide and 3 mg of α-casein or gelatin (Sigma)/ml. Concentrated conditioned medium was mixed with nonreducing Laemmli sample buffer and incubated at 37°C for 15 min. After electrophoresis, the gels were incubated for 1 h in 2.5% Triton X-100 at room temperature, followed by 24 to 48 h in substrate buffer (100 mM Tris-HCl [pH 7.4]–15 mM CaCl2) in the absence or presence of GM6001 (0.2 mM in DMSO; supplied by Glycomed Corporation and obtained from Z. Werb [12]), EDTA (10 mM), ortho-phenanthroline (1 mM in DMSO; Sigma), PMSF (5 mM), or AEBSF (0.5 mM; Calbiochem). Where appropriate, control gels were incubated with buffer containing solvent only. The gels were stained with Coomassie blue for 30 min and were destained with 30% methanol–10% acetic acid. Caseinase and gelatinase activities were visible as clear bands, indicative of proteolysis of the substrate protein.
Id-1 induces an invasive, migratory phenotype in mammary epithelial cells.SCp2 mammary epithelial cells grow as a monolayer in 5% serum. When given lactogenic hormones and basement membrane ECM in serum-free medium, they arrest growth, form three-dimensional alveolar structures, and express the milk protein β-casein (8). Alveoli formed by SCp2 cells are stable, maintaining their structure and β-casein expression for more than 2 weeks. Under these conditions, Id-1 is not expressed. By contrast, SCp2 cells that constitutively express Id-1 form poorly compacted alveoli that become increasingly disorganized; after 6 to 8 days, cells at the periphery detach from the structure and synthesize DNA (9).
Using a pooled population of SCp2 cells that constitutively express a murine Id-1 transgene (SCp2–Id-1 cells) (9), we more precisely monitored the fate of cells that detached from the alveolar structure. Within 10 days, approximately 30 to 40% of the SCp2–Id-1 alveolar structures showed substantial disintegration. Following detachment from the alveolar structure, SCp2–Id-1 cells actively invaded and migrated through the surrounding ECM (Fig.1). The migrating cells had an elongated nuclear morphology, compared to the rounded nuclei of cells in the early stages of disaggregation. Initial detachment and invasion occurred in the absence of cell proliferation (Fig. 1A). However, 2 to 4 days after initial detachment, SCp2–Id-1 cells that had migrated extensively through the ECM were abundant, and many of these cells synthesized DNA (Fig. 1B to D). For the most part, DNA synthesis was evident only in cells that had migrated some distance from the alveolar structure. Thus, the initiation of invasion and migration was not due to resumption of growth; rather, cells resumed proliferation only after they had detached and migrated from the three-dimensional structure. As previously described (9), spheres comprised of control cells transfected with the vector alone were very stable, remaining viable and morphologically unchanged even after more than 10 days on basement membrane ECM.
To quantify the invasion and migration of SCp2–Id-1 cells, they and control cells were assayed in Boyden chambers (2). Cells were added to the upper portion of the chamber; conditioned medium from mouse fibroblasts, used as a source of chemoattractants (2), was added to the lower compartment. The porous filter separating the two compartments was coated with basement membrane ECM. After a 16- to 20-h incubation, cells that had migrated through the ECM to the lower surface of the filter were fixed, stained, and counted (Fig.2). The 16- to 20-h incubation time ensured that only a small fraction of invasive cells migrated through the filter, which in turn ensured that the fraction of migratory cells was small enough to score reliably.
Four types of cells were compared in this assay: (i) parental SCp2 cells, (ii) SCp2 cells transfected with an insertless vector, (iii) SCp2–Id-1 cells, and (iv) SCp2 cells transfected with the Id-1 cDNA in the antisense orientation. Of these cell types, only SCp2–Id-1 cells were invasive. Under these assay conditions, none of the control (parental or insertless-vector) cells and none of the cells expressing antisense Id-1 migrated through the filter. By contrast, 0.7 to 1% of a population of one of the most invasive breast cancer cell lines (MDA-MB-231, previously described [43]) migrated through the filter, although only about 0.05% of the SCg6-transformed cells, which were previously shown to be invasive (28), migrated through the ECM to the lower surface of the filter (data not shown). In the case of the SCp2–Id-1 cells, 0.2 to 0.3% migrated through the filter. Thus, SCp2–Id-1 cells, which were transfected with a single gene, were 20 to 30% as invasive as one of the most aggressive breast cancer cell lines (which harbors multiple mutations) and four- to sixfold more invasive than their SCg6-transformed counterparts.
We conclude that constitutive expression of the Id-1 gene can induce an invasive and migratory phenotype in nontransformed and nontumorigenic SCp2 mammary epithelial cells.
Constitutive Id-1 expression is not sufficient for anchorage-independent growth or tumorigenicity.In many model systems of malignant transformation, unregulated expression of normal or activated proto-oncogenes drives cell proliferation, and invasiveness often develops subsequent to, or concomitant with, tumorigenicity. Although Id-1 did not appear in this regard to act like a typical oncogene, we nonetheless asked whether constitutive Id-1 expression transformed SCp2 cells, using the criteria of anchorage-independent growth and tumorigenicity in nude mice.
We first tested the ability of the cells to grow in an anchorage-independent manner. As expected, control cells and cells transfected with Id-1 in the antisense orientation failed to grow in soft agar (Fig. 3A and C). Similarly, SCp2–Id-1 cells failed to form colonies in soft agar, remaining as single cells for at least 14 days (Fig. 3B). It is interesting that, in soft agar, SCp2–Id-1 cells appeared twice as large as control cells; the reason for this size increase is not known. Malignant TCL1 cells (isolated from a murine mammary tumor [28]), used as a positive control, formed large colonies after 14 days in soft agar (Fig. 3D). We conclude that constitutive expression of Id-1 does not induce anchorage-independent growth in SCp2 mammary epithelial cells.
We next tested SCp2–Id-1 cells for their ability to form tumors. Cells were injected subcutaneously into nude mice. The positive control, TCL1 cells, formed tumors (at least 1 cm3) within 3 weeks (Table 1). The same was true for SCg6, a cell line with mesenchymal and transformed properties that was isolated from the same population from which SCp2 cells were isolated (8) (Table 1). By contrast, neither parental SCp2 cells, SCp2 cells expressing the Id-1 antisense cDNA, nor SCp2–Id-1 cells formed tumors after 5 months (Table 1).
We conclude that constitutive Id-1 expression in SCp2 mammary epithelial cells is not sufficient to lead to the transformed phenotypes of anchorage-independent growth in culture and in vivo, despite its ability to induce an invasive phenotype.
Isolation and characterization of cloned SCp2–Id-1 cells.The experiments described thus far used a pooled population of SCp2–Id-1 cells, which is heterogeneous with respect to Id-1 expression. To eliminate this heterogeneity and better define the role of Id-1 in inducing an invasive phenotype, we isolated single-cell-derived SCp2–Id-1 clones that expressed the Id-1 transgene to varying levels. The clones were assessed for cytokeratin filaments (a general characteristic of epithelial cells), morphology in monolayer culture, and ability to form alveolar structures in response to basement membrane ECM. In addition, RNA was isolated 5 days after the cells were exposed to basement membrane and hormones and was analyzed for Id-1 and β-casein mRNA. The Id-1 transgene mRNA was distinguishable from the endogenous Id-1 mRNA by its slightly larger size; the endogenous transcript was barely detectable under these conditions (9).
One subclone, SCp2–Id-1A cells, did not express detectable Id-1 transgene mRNA (Fig. 4a, lane 1). These cells grew as compact colonies in monolayer culture and expressed cytokeratin filaments (Fig. 4b, panel B). They also differentiated similarly to untransfected SCp2 cells, as judged by their ability to express high levels of β-casein mRNA (Fig. 4a, lane 1) and form stable alveolar structures (data not shown), like untransfected SCp2 cells. These cells were therefore used as negative controls.
SCp2–Id-1B and SCp2–Id-1C cells expressed moderate levels of the Id-1 transgene, which were below the levels of Id-1 mRNA expressed by proliferating control cells. These cells expressed lower levels of β-casein mRNA than SCp2–Id-1A cells (Fig. 4a, lanes 2 and 3), but they expressed cytokeratin filaments and formed alveolar structures (data not shown).
Finally, SCp2–Id-1D and SCp2–Id-1E cells expressed high levels of the Id-1 transgene and undetectable levels of β-casein (Fig. 4a, lanes 4 and 5). In monolayer culture, SCp2–Id-1E cells were less cuboidal and grew as more-dispersed entities than SCp2–Id-1A cells (Fig. 4b, panels C and D). Their failure to express β-casein was not due to a loss of epithelial characteristics. SCp2–Id-1E cells, which expressed the highest levels of Id-1, as well as SCp2–Id-1D cells (data not shown), expressed cytokeratin filaments (Fig. 4b, panel D). However, SCp2–Id-1D and SCp2–Id-1E cells, like the pooled SCp2–Id-1 cells, formed only loose alveolar structures, from which they eventually detached and invaded the ECM (see Fig. 7) (data not shown).
These results confirm in cloned populations that mammary epithelial cells constitutively expressing Id-1 do not undergo a complete epithelial-to-mesenchymal transition; they retain some epithelial-cell characteristics (such as keratin expression) but fail to functionally differentiate and to maintain three-dimensional organization on the ECM. SCp2–Id-1A and SCp2–Id-1E cells, which express undetectable and high levels of the Id-1 transgene, respectively, were used for further studies.
A potentially novel metalloproteinase is secreted by Id-1-expressing cells.The ability of SCp2–Id-1 cells to invade the ECM suggested that Id-1 might induce expression of ECM-degrading proteases. The major classes of proteases that degrade ECM are serine, cysteine, and aspartyl proteases, and metalloproteinases (10). The Zn2+-containing, Ca2+-stabilized MMPs are of particular interest because they are implicated in the remodeling of the mammary gland during involution (29, 41) and the initial steps of tumor-cell invasion (25). Of the major MMPs, interstitial collagenase (56 kDa) and gelatinases A (72 kDa) and B (92 kDa) are detectable on gelatin substrate gels, whereas stromelysins (57 kDa for stromelysin-1) and matrilysin (30 kDa) are detectable on casein substrate gels (reviewed by Fisher and Werb [10]; see also reference 22).
We examined the secretion of proteases by SCp2–Id-1A and SCp2–Id-1E cells, using conditioned medium and gelatin or casein substrate gel zymography. Cells were incubated in serum-free medium for 3 days prior to collection of conditioned medium for zymography. Under these conditions, the endogenous Id-1 gene is not expressed (9), and SCp2–Id-1A and SCp2–Id-1E express undetectable and high levels of the Id-1 transgene, respectively.
Gelatin substrate gels showed that SCp2–Id-1A and SCp2–Id-1E cells differed only in the secretion of a high-molecular-mass (approximately 120-kDa) gelatinase. The 120-kDa gelatinase was abundantly expressed by serum-deprived SCp2–Id-1E cells (Fig.5A, lane 2) as well as SCp2–Id-1D cells (data not shown). Secretion of this 120-kDa gelatinase was not due to clonal variation. Conditioned medium from the uncloned SCp2–Id-1 pooled population also showed a gelatinase of ∼120 kDa (Fig.5C, lane 2). This gelatinase was undetectable in serum-deprived SCp2–Id-1A (Fig. 5A, lane 1) and control SCp2 (Fig. 5C, lane 1) cells. Thus, secretion of a 120-kDa gelatinase correlated with Id-1 expression.
Secretion of the 120-kDa gelatinase correlated with expression of the endogenous Id-1 gene as well as with that of the Id-1 transgene. Thus, the 120-kDa proteinase was secreted by SCp2–Id-1A cells (in which expression of the Id-1 transgene is undetectable) while they were proliferating in monolayer culture (Fig. 5B, lane 2). Under these conditions, the endogenous Id-1 gene is expressed at high levels (9).
The 120-kDa gelatinase had characteristics of an MMP. It was sensitive to the MMP inhibitors GM6001 (Fig. 5D, lane 4), EDTA, andortho-phenanthroline (data not shown). By contrast, it was insensitive to the serine protease inhibitors PMSF (Fig. 5D, lane 6) and AEBSF (data not shown). The 120-kDa MMP appeared to be the only MMP secreted by Id-1-expressing cells. The two gelatinases with apparent molecular sizes of 70 and 90 kDa, which were expressed by both SCp2–Id-1A and SCp2–Id-1E cells, were not inhibited by any of the MMP inhibitors GM6001 (Fig. 5D, lane 4), ortho-phenanthroline, and EDTA (data not shown), and therefore neither is likely to be gelatinase A or B.
Casein substrate gels showed one major caseinase of approximately 50 kDa that was expressed by both SCp2–Id-1A and SCp2–Id-1E cells. This protease was not inhibited by the metalloproteinase inhibitor GM6001 (Fig. 5E). Therefore, it is most likely not the metalloproteinase stromelysin-1.
To definitively rule out the possibility that gelatinases A and B were expressed in SCp2 cells, as well as the possibility that the 120-kDa MMP was a complex between gelatinase B and its carrier protein (21), we analyzed RNA by PCR and Southern blotting for gelatinase-A and -B mRNAs. SCp2–Id-1A cells, which do not express the Id-1 transgene, SCp2–Id-1E cells, which express high levels of the Id-1 transgene, and the mesenchyme-like mammary SCg6 cells were deprived of serum for 3 days before RNA was extracted and synthesized into cDNA for PCR analysis (Fig. 6). The 326-bp PCR product expected from the gelatinase-A cDNA and the 190-bp product expected for the gelatinase-B cDNA were detected only in SCg6 cells (Fig. 6, lane 3). We conclude that SCp2 cells, whether or not they express Id-1, do not express gelatinase A or B and that therefore the 120-kDa gelatinase is not a gelatinase B-containing complex (21).
We conclude that SCp2 mammary epithelial cells secrete a single detectable MMP, having an apparent molecular size of 120 kDa, when they express Id-1. This MMP does not belong to the stromelysin subclass of MMPs, which degrades casein, but rather is a type IV collagenase MMP family member and thus degrades gelatin, a denatured collagen.
The Id-1-related MMP is essential for the invasive phenotype of SCp2 cells.Because the 120-kDa MMP appears to be the only proteinase whose secretion correlates with Id-1 expression, and constitutive Id-1 expression renders cells invasive, we explored the possibility that this MMP is critical for the invasive phenotype of mammary epithelial cells.
We first tested the abilities of SCp2–Id-1A and SCp2–Id-1E cells to invade basement membrane ECM in a Boyden chamber invasion assay (Fig.7). SCp2–Id-1A cells, like untransfected SCp2 cells (Fig. 2), were not invasive, or only minimally invasive, in this assay (Fig. 7). Under the assay conditions, the endogenous Id-1 gene is not expressed and SCp2–Id-1A cells express undetectable levels of the Id-1 transgene. By contrast, SCp2–Id-1E cells, like uncloned SCp2–Id-1 cells (Fig. 2), were demonstrably invasive under the same conditions, consistent with the high levels of the Id-1 transgene expressed by these cells.
To test the role of the 120-kDa MMP in the invasive phenotype induced by Id-1, we used MMP inhibitors in the invasion assay. We first tested the toxicities of two compounds, GM6001 and phenanthroline. SCp2 cells were treated with either compound, the solvent (DMSO), or nothing for the duration of the invasion assays (20 h), and viability was assessed by trypan blue exclusion. There were no differences in viability among untreated, DMSO-treated, and GM6001-treated cells (data not shown). By contrast, all the phenanthroline-treated cells died within 20 h of treatment. We therefore used GM6001 in the invasion assay. GM6001 reduced the invasiveness of SCp2–Id-1E cells about fourfold (Fig. 7, lane 3). Because the 120-kDa gelatinase is the only detectable MMP expressed by these cells, this result suggests that much of the invasive phenotype induced by Id-1 can be attributed to the 120-kDa MMP.
Id-1 and the 120-kDa gelatinase are expressed during mammary gland involution.In studying proteases during mouse mammary-gland development, Talhouk et al. (41) described a gelatinase having a molecular size greater than 110 kDa that was not expressed during lactation (Fig. 8, top panel, lane 1) but was expressed during the early stages of involution (days 1 and 2 [lanes 2 and 3], declining by day 3 [lane 4]). The identity or function of this gelatinase was not determined or discussed. To explore the possibility that this gelatin-degrading proteinase may be the 120-kDa MMP expressed by Id-1-expressing cells, we isolated RNA from lactating and involuting mouse mammary glands and measured Id-1 expression by Northern analysis (Fig. 8, lower panels). Id-1 mRNA was undetectable in the lactating gland (lane 1) but was highly expressed early in involution (day 1 and 2 [lanes 2 and 3]); Id-1 expression began to decline by the 3rd day of involution (lane 4). Thus, the correlation between the expression of Id-1 and a 120-kDa gelatinase observed in mammary epithelial-cell cultures is also seen in the intact mammary gland during involution.
Id-1 and 120-kDa gelatinase expression in invasive human breast cancer cells.Our finding that ectopic Id-1 expression induced a 120-kDa gelatinase and an invasive phenotype in mouse mammary epithelial cells suggested that Id-1 and its associated gelatinase could, at least in some instances, contribute to human breast cancer progression. To begin to explore this possibility, we examined human breast cancer cell lines exhibiting varying degrees of invasiveness in culture and in vivo.
We examined four differentiated, essentially noninvasive breast cancer cell lines, T47D, MCF-7, ZR75-1, and SKBR-3 (43), and five poorly differentiated and invasive cell lines, Hs578T, BT-549, MDA-MB-231, MDA-MB-436, and MDA-MB-435 (27, 30, 43, 50) (Fig. 9). These cell lines have been evaluated for invasiveness in culture, by using the Boyden chamber assay (2), and in vivo, by using metastatic tumor formation in nude mice (43). By both assays (under serum-free and/or estrogen-free conditions), T47D, MCF-7, ZR75-1, and SKBR-3 cells were noninvasive. By contrast, Hs578T, BT-549, and particularly MDA-MB-231, MDA-MB-436, and MDA-MB-435 cells were highly invasive by both assays. We confirmed the reported invasive potentials of these cells, using the Boyden chamber assay (data not shown).
When cells were cultured in serum-free medium for 2 days, Id-1 mRNA was undetectable in the noninvasive T47D, MCF-7, ZR75-1, and SKBR-3 cells (Fig. 9, lanes 1, 2, 6, and 7) but was easily detectable in the highly invasive MDA-MB-231, MDA-MB-436, and MDA-MB-435 cells (Fig. 9, lanes 5, 8, and 9). Of the other invasive cells, Hs578T expressed low levels of Id-1 mRNA (Fig. 9, lane 3), whereas Id-1 mRNA was undetectable in BT-549 (Fig. 9, lane 4). Thus, the invasive potential of the human breast cancer cell lines MDA-MB-231, MDA-MB-436, MDA-MB-435, and, to a lesser extent, Hs578T could, at least in part, derive from unregulated expression of Id-1 and its associated 120-kDa gelatinase.
Consistent with this idea, a 120-kDa gelatinase was detected in conditioned media from the invasive cells that expressed Id-1 (Hs578T, MDA-MB-231, MDA-MB-436, and MDA-MB-435; Fig.10, lanes 3, 5, 8, and 9, respectively). This gelatinase was not detected in conditioned media from the noninvasive cell lines T47D, MCF-7, ZR75-1, and SKBR-3 (Fig.10, lanes 1, 2, 6, and 7) or from the invasive cell line that did not express Id-1 (BT-549; Fig. 10, lane 4). The 120-kDa gelatinase expressed by the human breast cancer cells comigrated with the 120-kDa gelatinase expressed by Id-1-transfected SCp2 cells (Fig. 10, lane C). As previously reported (1), the 72- and/or 92-kDa gelatinases were detected in most of these human cell lines, whether or not they were invasive. Despite the secretion of these gelatinases by the cells, only the 120-kDa gelatinase-expressing cells were invasive in the Boyden chamber invasion assay (reference 43and data not shown). The exception was the invasive BT-549 cell line, which neither expressed Id-1 mRNA nor secreted the 120-kDa gelatinase. BT-549 cells express many MMPs (by zymography), including high levels of membrane type 1 MMPs (11).
Thus, among nine human breast tumor cells examined, only Id-1-expressing cells also expressed the 120-kDa gelatinase, and all Id-1-negative cells failed to express the 120-kDa gelatinase. Moreover, the Id-1- and 120-kDa gelatinase-expressing cells were all invasive in culture and in vivo.
The mammary gland is one of the few organs that undergo striking morphological and functional changes during adult life, particularly during pregnancy, lactation, and involution. In both humans and mice, fetal, virgin adult, and pregnant mammary glands undergo extensive temporal and spatial remodeling, which entails invasion, migration, and relocation of cells to generate the ductal and alveolar structures of the gland. Once lactation is terminated, there is additional and extensive tissue remodeling as the gland returns to its resting state. In recent years, progress has been made in elucidating the mechanisms that regulate mammary gland-specific gene expression and the transformation of mammary epithelial cells to malignancy (3, 39). However, much less is known about the mechanisms, particularly the transcriptional mechanisms, that regulate the development and remodeling of the normal mammary gland.
SCp2 cells as a model for normal mammary epithelial cells.SCp2 is an immortal murine cell line that nonetheless expresses many characteristics of epithelial cells in the pregnant and lactating mammary gland. SCp2 cells proliferate in monolayer culture in response to serum growth factors but arrest growth, form alveolar structures, and express milk proteins in response to lactogenic hormones and basement membrane components. Arrested growth is necessary, but not sufficient, for differentiation. The differentiation of SCp2 cells in culture is remarkably similar to the differentiation of mammary epithelial cells in vivo (8). Here, we extend this similarity to expression of a 120-kDa MMP that appears to be controlled by Id-1, a negative regulator of bHLH transcription factors (4).
Id-1 as a negative regulator of mammary epithelial-cell differentiation.During proliferation, but not during arrested growth or differentiation, SCp2 cells express Id-1. The expression of Id-1 and that of the milk protein β-casein are inversely correlated in cultured SCp2 cells (9), as well as in the mammary gland in virgin, pregnant, and lactating mice (9a). Indeed, Id-1 is a negative regulator of the functional differentiation of SCp2 cells. When constitutively expressed, Id-1 prevents the strong cell-cell contacts typical of differentiated cells and blocks milk protein expression. Although the precise mechanism by which Id-1 inhibits differentiation is not known, it is clear that it does not act by preventing the growth arrest induced by hormones and ECM (9).
Id-1 is presumed to repress differentiation by inhibiting one or more bHLH transcription factors. By analogy with the role of bHLH proteins in the differentiation of muscle, neuronal, and lymphoid cells (24, 40, 47), bHLH transcription factors may be required for differentiation-specific gene expression in the mammary gland. However, our results suggest an additional role for bHLH proteins in the mammary gland: repression of a 120-kDa MMP, whose activity permits the epithelial cells to migrate and invade the ECM.
An Id-1-regulated gelatinase expressed by mammary epithelial cells.Id-1 expression, whether originating from the endogenous gene or a transgene, correlated strongly with the expression of a 120-kDa gelatinase having the characteristics of an MMP. This protease appeared to be the only metalloproteinase expressed by SCp2 mammary epithelial cells. The well-characterized MMPs stromelysin and gelatinases A and B (72- and 92-kDa type IV collagenases) were not expressed by SCp2 cells. By contrast, gelatinases A and B were expressed by SCg6, a stroma-like cell line derived from the same culture from which SCp2 cells were cloned (8). SCg6 cells also express stromelysin-1 (28). These findings suggest that the expression of stromelysin and gelatinases A and B during involution of the mammary gland may derive from the nonepithelial cells in the tissue (29).
The epithelial cells of the mammary gland, on the other hand, may express the 120-kDa MMP. Talhouk et al. (41) described a gelatinase with an apparent molecular size exceeding 110 kDa that was expressed during the early stages of involution. We found that Id-1 mRNA was not expressed during lactation, when the 120-kDa gelatinase is undetectable, but was expressed early in involution (days 1 and 2). We suggest that this gelatinase may be the 120-kDa MMP identified in Id-1-expressing SCp2 cells. Thus, there is a correlation between Id-1 expression and secretion of a 120-kDa gelatinase in vivo, as well as in cultured cells.
The Id-1-regulated gelatinase is critical for epithelial-cell invasiveness.SCp2 cells arrest growth when in contact with basement membrane ECM. Under these conditions, Id-1 is not expressed, the cells maintain strong contacts, and they do not invade the surrounding ECM (9). Constitutive Id-1 expression did not prevent the growth arrest but conferred an invasive phenotype on the cells. Only after Id-1-expressing SCp2 cells had invaded the ECM did they resume proliferation. Thus, Id-1 appeared to be a regulator of the invasive phenotype rather than a stimulator of cell proliferation per se. This invasive phenotype, in turn, appeared to depend primarily on the 120-kDa gelatinase (MMP). This MMP was the only detectable target of GM6001, a nontoxic MMP inhibitor (12), and GM6001 effectively inhibited the invasive phenotype of Id-1-expressing cells. Thus, Id-1 and its related 120-kDa MMP were key regulators of the invasive phenotype of SCp2 cells. During involution, the Id-1-associated MMP may participate in remodeling the gland in vivo. We suggest that Id-1 and its related MMP may be key regulators of the transient invasive phenotype acquired by the epithelial cells during certain stages of normal mammary-gland development and remodeling.
Id-1 and the 120-kDa gelatinase in tumor cell invasion.The invasive phenotype induced by Id-1 was not the result of malignant transformation. Id-1-expressing SCp2 cells did not grow in an anchorage-independent manner and did not form detectable tumors in nude mice. Thus, Id-1 differs from oncogenes such as v-Ha-ras, which converts mouse mammary epithelial cells into invasive but also tumorigenic cells (13). Furthermore, Id-1 did not induce an invasive phenotype by converting cells to a stromal or mesenchymal phenotype. Id-1-expressing SCp2 cells maintained their epithelial characteristics, such as keratin expression, and did not express stromal MMPs. Thus, the action of Id-1 differs from that of genes of the ets family. c-Ets, a transcription factor expressed by stromal fibroblasts, promotes epithelial tumor cell invasion (48) by inducing stromal MMPs such as stromelysin-1. E1AF, a new member of the ets family, induces an invasive and migratory phenotype in human MCF-7 breast cancer cells (19), presumably by inducing gelatinase B as well as stromelysin-1.
Although the Id-1-induced invasive phenotype was not a consequence of malignant transformation, our results with human breast cancer cells suggest that constitutive Id-1 expression, and its associated 120-kDa gelatinase, may play a role in the invasive phenotype of at least some aggressive human breast tumors. We hypothesize that Id-1 and the 120-kDa gelatinase may constitute a thus far unrecognized pathway for tumor cell invasion. A very recent report (32) suggests that the Id-1–120-kDa gelatinase pathway we describe here may be of substantial clinical importance. In that report, a gelatinase of approximately the same size as the one described here was detected in urine from metastatic breast cancer patients but not in urine specimens from patients with other types of cancer. The authors acknowledge that the identity of this gelatinase is as yet unknown but suggest that it might serve as a predictor of metastatic breast cancer. By contrast, the 72- and 92-kDa gelatinases detected in urine were suggested to serve as predictors of organ-confined cancers. These suggestions are consistent with our results showing that the 72- and/or 92-kDa gelatinase is expressed by differentiated and noninvasive human breast cancer cells, whereas the 120-kDa gelatinase is expressed only in invasive breast cancer cells.
In conclusion, we propose that Id-1 regulates the invasive phenotype of breast epithelial cells, in part through the activity of a 120-kDa gelatinase, during normal mammary-gland development and remodeling. Although this phenotype is not necessarily linked to tumorigenesis, it may well be reactivated during progression toward malignancy in some breast cancers, for example, during the transition from an in situ to an invasive carcinoma. We do not yet know whether Id-1 induces the 120-kDa gelatinase by directly inactivating a bHLH repressor of the gene or whether it acts indirectly by altering the expression of other genes. We are currently attempting to clone the 120-kDa gelatinase in order to answer these questions.
We thank Y. Jen (Memorial Sloan-Kettering Cancer Center, New York, N.Y.) for the murine Id-1 cDNA, J. Rosen for the β-casein cDNA, and Z. Werb for the gelatinase-A and -B cDNAs and the GM6001. We also thank S. Liang, A. Lochter, R. Lupu, and Z. Werb for assistance with some of the experiments and for helpful discussions.
P.-Y.D. and C.Q.L. contributed equally to this work.
This work was supported by grants from the U.S. Department of Energy (contract DE-AC03-76SF00098) to M.J.B. and J.C. and by a New Investigator Award from the University of California Breast Cancer Research Program (1KB0274) to P.-Y.D.
- Received 20 October 1997.
- Returned for modification 16 December 1997.
- Accepted 21 May 1998.
- Copyright © 1998 American Society for Microbiology


text:
PLEASE VISIT Greenspan410.com
PLEASE VISIT GREENSPAN410.COM - Add GREENSPAN Radio Station on Pandora || Follow @Greenspan410 ||
Thursday, August 23, 2012
Tuesday, July 3, 2012
New Music: "Str8up" prod. Che Fever
Follow @Greenspan410 on Twitter! Download "Str8 Up"
Wednesday, June 13, 2012
June 17th... @PeterHadar x @Shyvonne x @Greenspan410 Live @ EMP Collective
Friday, May 25, 2012
Monday, May 14, 2012
Thursday, May 3, 2012
Sunday, April 8, 2012
Tuesday, April 3, 2012
Thursday, March 22, 2012
Sunday, March 18, 2012
Saturday, March 17, 2012
Kool G. Rap - Fast Life
Kool G. Rap - Fast Life [feat. Nas]
Wednesday, March 14, 2012
Tuesday, March 13, 2012
Video: Greenspan - "Ride" feat Nephew
Video: Greenspan - "Ride" feat Nephew
Dir. @FeverFilms410
Co-dir/Pro. - @_Boosta
Dir. @FeverFilms410
Co-dir/Pro. - @_Boosta
Tuesday, March 6, 2012
Sunday, January 29, 2012
Greenspan x Cabal
Images by Chinwe Click to check out more
Thursday, January 26, 2012
Earbuds w/ @AlGreat11 & @Greenspan410
Come to the Ear Buds Listening party for,
Al Great and Greenspan
Aesthetics Boutique
6400 Fredrick Rd
Catonsville, MD
@Irockfox will have Fall/Winter product for sale at the event. Come through and get what you need for the season.
For more information,
Visit www.irockfox.com
Follow @IROCKFOX
Also, @NewVintagebySam will displaying Jewelry!!! for more info --> @ShesoGlamorous
Thursday, December 22, 2011
Over the weekend...
Over the weekend, @Greenspan410 x @Feevaleo x @JonJonSinnati shooting videos and making music in Baltimore
Saturday, November 12, 2011
Archive: @Greenspan410 - "Searching" [Roy Ayers]
From Greenspan 2008 release GOT GREEN
Subscribe to: Posts (Atom)


text:
Bavarian Lakes Cycling Holiday
CategoryCycle Touring
Self Guided
Duration/s6 or 7 nights
Start / FinishMunich / Munich
Distance190 miles/305 kms
Tour code014-105CDE
A lazy loop ride that leads you south from Munich to Garmish Partenkirchen near the Austrian border and back again. Leisurely cycling through a region dotted with idyllic lakes, fairytale villages and jaw-dropping mountain scenery. See Murnau am Staffelsee, Garmish Partenkirchen, Wallgau, Bad Tölz and Tegernsee.
Munich is a vibrant city with much to see and do. Visit the famous Hofbraühaus to enjoy a frothy litre mug of beer while swaying to the Oom-Pah music with the locals. The City Museum of Munich offers a fascinating insight into the diverse history of the city and we also highly recommend a stroll through the wonderful Englischer Garten – a large park area in Schwabing where you can enjoy a beer and wurst in the fresh air.
Leaving the city you'll enter a quieter world, cycling along quiet lanes past clear lakes to colourful, baroque towns and rustic villages. Murnau, a lovely old market town, enjoying an idyllic location on the shore of Lake Staffelsee is lovely place to spend the night. In Garmish-Partenkirchen we highly recommend taking the mountain railway up to a dizzying 3000 meters for some amazing views of the surrounding alps.
A lovely day's riding through quiet valleys will bring you to Wallgau, a charming town where Bavarian traditions are still very evident and part of everyday life. In Bad Tölz you can wander along the bustling 'Tölzer' street admiring the baroque houses, perhaps taking a seat in one of the many cafes, bars or restaurants for a spot of people watching.
Spectacular lake views await you at Tegernsee Lake before a final day's ride leads you gently back to the Bavarian Capital.
This cycle route follows easy, flat cycle paths and back roads. There are a few unpaved sections and some minor climbs to contend with and only a few very short sections on roads with more traffic, but the route is well within the reach of most leisure cyclists.
Itinerary & Map
The itinerary below describes the 7 night tour. If you opt for the shorter 6 night trip the itinerary is the same except that day 6 will be omitted. If you choose the longer 8 night trip you will break the journey on day 2 and stay in Lake Starnberg before cycling on to Murnau am Staffelsee on day 3. This cycle route follows easy, flat cycle paths and back roads. There are a few unpaved sections and some minor climbs to contend with and only a few very short sections on roads with more traffic, but the route is well within the reach of most leisure cyclists.
Day 1: Arrive Munich
On arrival make your way to your first hotel in Munich. After check-in you're free to explore the Bavarian capital. If you've got time, a stroll in the Englischer Garten is highly recommended, but whatever you do, don't miss the opportunity to join the locals in one of the city's world-famous beer gardens. German sausages and salty pretzels washed down with a stein of frothy local beer in the fresh air – lovely!
Day 2: Munich – Murnau am Staffelsee, 41 miles/65 kms
Leaving Munich, your cycle tour commences with a ride south through Forstenrieder Park to Starnbergersee. This substantial lake is not far from the city but feels a world away. Follow the lakeside road to Seeshaupt at the very southernmost point of the lake then pass through typical Bavarian towns and villages and visit lakes Ostersee and Riegsee en route to the market town of Murnau am Staffelsee, on the edge of the Bavarian Alps.
Day 3: Murnau am Staffelsee – Garmisch Partenkirchen, 25 miles/40 kms
First you cycle through Murnauer Moos – central Europe's largest swampland – before continuing alongside the River Loisach towards the majestic Alps and the village of Garmisch Partenkirchen. You'll spend the night in Garmish and today's ride is shorter than most, to give you time to take the mountain railway to almost 3,000m, where you'll be treated to a panoramic Alpine view from the Zugspitz Mountain.
Day 4: Garmisch Partenkirchen – Wallgau, 28 miles/45 kms
You'll start the day with a train ride to Scharnitz. Then saddle up and cycle uphill through gorges and meadows to the source of the River Isar. From there you'll be following a valley track with views stretching across the mountains, which will take you to Wallgau, a charming small town in the very south of Bavarian where local traditions are still kept alive.
Day 5: Wallgau – Bad Tölz, 31 miles/50 kms
Cycling through the Isarwinkel area of Upper Bavaria you arrive at idyllic Lake Sylvenstein, surrounded by snow-capped mountains. There's plenty more wonderful scenery today as you approach Bad Tölz, a fine old town on the Isar boasting many ornate mansions and grand buildings and your home for the next two nights.
Day 6: Bad Tölz – Tegernsee – Bad Tölz, 28 miles/45 kms
This day is omitted if you opt for the 6 night version of the tour
It's a circular route today, heading out from Bad Tölz to visit more Bavarian countryside. Ride to Gmund on Lake Tegernsee, for some of the best views of the lake. Return to Bad Tölz via the Gaißbach valley. Enjoy a stroll along the colourful Tölzer Market Street.
Day 7: Bad Tölz – Munich, 38 miles/60 kms
Leave Bad Tölz behind to follow the Isar to the thickly forested Grünwald, and you'll be almost back in Munich. There will be a couple of short climbs to tackle en route to the Bavarian capital, where you'll have more time to get to know one of Germany's most appealing cities. Its museums are world-class, and it's also a city that's very easy and safe to make your way round by bike.
Day 8: Munich
After breakfast it's time to check-out and head for home or your next adventure.
Dates & Prices
|2021 Arrival Dates
|Arrivals daily from 12 April to 04 September
Scroll/swipe to see more
|2021 Prices
Per person
03 Apr
30 Apr
01 May
11 Jun
12 Jun
04 Sep
|6 Nights
|Superior category
|7 Nights
|Superior category
Scroll/swipe to see more
|Deposit per person
Price Includes
6 or 7 x Buffet Breakfast
Luggage transfer (Unlimited, max weight 20kg per piece)
Maps & route information
Welcome briefing
Train Garmisch Partenkirchen - Scharnitz (excluding bikes)
GPS route data
Local assistance
Price Excludes
Bike hire (see supplements)
Helmet (€35, payable locally)
Bike ticket on train Garmisch Partenkirchen - Scharnitz (approximately €5)
Tourist tax (€2pppn, payable locally)
Half board: 7 x dinner (7 nights) (£209)
Extra night Munich, double/twin, superior (£84)
Extra night Munich, single, superior (£146)
Regular bike with pannier & lock. See practical information (£75)
Regular Plus bike with pannier & lock (£136)
Electric bike with pannier & lock (£198)
50% for children 6 -11 years sharing with 2 adults
25% for children 12 -14 years sharing with 2 adults
Practical Information
On this tour you can choose between a regular bike, a regular Plus bike or an electric bike.
Regular bikes provided are modern alloy framed models with 21 speeds. The bikes come equipped with pannier, lock and a repair kit with pump.
The regular Plus bike offers a step up in terms of components (hydraulic Magura brakes HS 11, Shimano Deore XT) and the frame provides a more sporty riding position. High quality, suntour suspension forks ensure a comfortable ride on these bikes. The bikes come equipped with pannier, lock and a repair kit with pump.
Electric bikes are also available.
We suggest you bring your own helmet for safety and hygiene reasons. Alternatively, helmets are available to buy (payable locally, €35). This must be advised at the time of booking.
Below you will find examples of the types of accommodation typically used on this tour. Please note that the hotels we book for you may differ slightly from those described but they will certainly be of a similar standard.
Superior: Good quality hotels and guesthouses.
Munich: Sheraton Westpark
Murnau: Hotel Angerbräu
Garmisch: Hotel Vier Jahreszeiten
Wallgau: Posthotel Wallgau
Bad Tölz: Kolberbräu
Local taxes
Some hotels have started to charge a local/tourist tax. These taxes are not included in the sale price of the tour and must be paid directly by you at check-in or checkout at the hotels. This fee varies and is ever changing but as a guide, you may be asked to pay between €1-€4 per person per night
Getting There
Fly to Munich
- Date - 20/06/2019
- Name - Pat McIntyre
- Service rating - 5.0
- Service comment - Ally who I dealt with us friendly and helpful and dealt with everything as I would have expected.
- Product rating - 5.0
- Product comment - The route was beautiful. The agent from Eurobikes was very good and we were assured that if there was any issue on the trip he was only a phone call away. I took my own bicycle seat and put it on for me and adjusted it to suit. The map and instructions were very clear. There were only a couple of miss prints in the written instructions (turn left which should have been right) but because the route was marked on lamposts at every change of direction it was easy to spot the error and proceed in the correct direction. I would highly recommend this trip to any one who is not the fittest. It was not too hard and when it got a bit steep we enjoyed a few hundred yards walking. We had three days rain but it did not spoil our enjoyment. If anything the low hanging clouds made the landscape even more photogenic.
- Flexitreks response - Thanks Pat. Rain is never welcome on cycling holidays but you manage to put a positive spin on it which is great! I find those lamppost directional stickers work really well and save me from having to look at the route notes quite so often. Love the moody photo by the way!
- Date - 26/09/2018
- Name - David Green
- Service rating - 5.0
- Service comment - Very well managed holiday. The bicycle handover was thorough and the routes explained competently.
- Product rating - 5.0
- Product comment - All the hotels were very good, the maps, guidance notes and route marking were excellent.
- Flexitreks response - Service rating : Many thanks for the 5 star review David - we really appreciate it.
Product : Excellent, just as it should be and thank you!
- Date - 27/06/2017
- Name - Anonymous
- Service rating - 4.0
- Service comment - Kept us informed when there was uncertainty over the booking
- Product rating - 4.0
- Product comment - Organisation - good.
Way marking/maps/directions - very good
Bikes - disappointing (too small and HEAVY)
Hotels - a mixed bag from excellent to OK
- Flexitreks response - Many thanks for the review. We were disappointed that you found the bikes to be too small and heavy. Measurements provided by our customers are used to calculate frame sizes and this process has until now been 100% accurate. The bikes used on these tours are specifically designed and built for the job in hand and while there are lighter models available, the frames used provide the best combination of strength, durability and weight.
- Date - 20/06/2016
- Name - Bob
- Service rating - 4.0
- Service comment - Generally very good- we were kept well informed at the time of booking and a couple of weeks prior to the trip.
- Product rating - 5.0
- Product comment - The, route, itinerary, hotels and food were generally very good - the hotel in Wallgau stands out. The changing scenery and road surface varying from Tarmac to gravel made the trip very interesting and enjoyable. Our only problem was gear change issues with some of the bikes in our party of nine and some problems communicating with Eurobikes - but nothing that wasn't solvable.
- Flexitreks response - Many thanks for the review Bob, We're glad you and your group enjoyed your trip with us.
- Date - 13/06/2016
- Name - Jenny
- Service rating - 4.0
- Service comment - The service agent from Euro bikes was unclear with instructions. He found it difficult to speak English.
- Product rating - 4.0
- Product comment - The ride was great although a lot of the roads/ cycle ways were gravel and some quite rocky. This made it difficult to enjoy the scenery while looking to avoid the uneven track. This was for about 1/4 of the total ride. The forests and countryside were beautiful but some roads challenging
- Flexitreks response - Hi Jenny. Appreciate your point about the uneven tracks however sometimes these are the only realistic option as the alternatives are less scenic or involve busy roads. Glad you enjoyed the holiday overall.


text:
What a great show!
It was an honour and a pleasure for us to be involved in the Canadian Folk Music Awards Gala. Congratulations to all the nominees and winners, and congratulations to the inimitable Bill Garrett for putting together a wonderful show. We enjoyed every minute!
- Canadian Folk Music Awards
- Partridgeberry Jam and Bakeapple Ballads starting Jan 23!


text:
You have no items in your Shopping Basket.
Product details
On-trend comfort
Breeze through busy days with our stylish, soft-touch casual. Embracing a key trend the fashionable cup sole gives it a cool, contemporary look. The lace adjusts to fit a range of swelling while the cushioned footbed feels exceptionally comfy underfoot.
Why you'll love Kama...
- Deep, seam-free toe area is ideal for problem toes
- Cushioned footbed can be removed to fit an orthotic
- Sole is lightweight, durable and flexible
- Lace can be adjusted to accommodate changes in swelling
- Supportive around the heel
Care Guide
- Allow any mud or stains to dry before attempting to clean them. Once dry, remove any dirt with a soft brush and then wipe with a damp cloth.
- If your footwear gets wet allow it to dry naturally. Don't put them in the airing cupboard or near a fire or radiator. Stuffing them with newspaper or tissue will help ensure they maintain their shape whilst drying.
- This footwear should not be hand or machine washed.
Product features
Suitable for...
This trendy lace up is so much deeper and roomier than it looks due to the sole that wraps around it. The lace up gives good support and adjustment and will fit a range of foot shapes and levels of swelling.
Feefo reviews
We've partnered with Feefo, to help us collect genuine customer reviews. After you have received your order from us you will receive an email asking you for feedback about your purchase. Giving this feedback need take no more than 15 seconds.
Delivery & returns
Our standard UK delivery charge is £4.99 per order.
If the item is in stock we aim to despatch it within 24 hours although occasionally it may take up to 48 hours. We don't despatch at weekends or on public holidays. Delivery is via Royal Mail and usually takes 1 to 4 working days from despatch.
Next Working Day, Convenient Collect and Overseas Delivery are available on this item. Please click here for details.
For non-UK customers: Information on cost and delivery estimates are shown in the checkout.
This product is covered by our "No Quibble Money Back Guarantee" so you can return it to us for free (within the UK) for an immediate exchange or refund. Please click here for further details.


text:
The 3 PM reading, Playbill.com has learned, will boast the talents of James Ludwig (Jimmy Morgan), Michael Minarik (Munzer/Babyface Nelson), Bradley Dean (John Dillinger), Brooke Sunny Moriber (Polly), Megan Sikora (Anna), Jennifer Prescott (Woman in Bank/Helen), Mark Lotito (Teller/Purvis), William Parry (Bank Official/Hoover), Eric Jordan Young (Herbert Youngblood), Eric Morris (John "Red" Hamilton/G-Man) and Deborah Leamy (Jean). Samantha Anderson Ives is the show's Narrator; Todd Stern and Liz Kimball are featured in the ensemble. Dillinger features a book by Joel Gross and a score by Paul Adelman. Michael Unger will direct the reading with musical direction by Phil Reno. Rebecca Fleming is the stage manager.
The musical, according to press notes, is described as such: "Jimmy Morgan, a young reporter in Depression-era Chicago, goes undercover with John Dillinger, the iconic gangster, and succumbs to his corrupting influence. Attracted and repelled by Dillinger, Jimmy finds himself lost between two worlds, moral bearings unglued, ambition leading him into fatal compromise. Worse, Jimmy falls in love with Dillinger's girl, Polly, never realizing that it is Dillinger himself who is pulling the strings of their love triangle; all part of the gangster's plan to use Jimmy's life as a ticket to freedom."
The York Theatre Company is located in Manhattan at 54th Street just east of Lexington Avenue. Reservations for the free reading may be made by emailing firstname.lastname@example.com or by calling (212) 935-5824, ext. 24.
For more information visit www.yorktheatre.org.


text:
A cheap, sloppily edited, and monumentally dull adaptation that requires the audience to endure scene after scene of indecipherable corporate lingo. Full review by Chris Pandolfi.
FilmWatch is a community of awesome people posting and discussing the latest movie news. It's part of NewsBoiler, a network of social news sites covering today's pop culture.


text:
The Hon. Bryon Wilfert, P.C.
Bryon Wilfert brings 26 years of experience in elected political office both as a municipal councillor and a Member of Parliament. Bryon brings a tremendous range of experiences at both levels of government.
Bryon's years of experience give him an unparalleled perspective with a rational and proactive approach to problem-solving. He is capable of making fair and knowledgeable assessments particularly based on his time as an international consensus-builder. Over the years, Bryon has built up relationships in countries such as Peru, Vietnam and Nigeria. His understanding of the importance of trade relations allows him to access more opportunities.
Known for his strong leadership skills, untiring work ethic and his balanced approach to issue management, Bryon has also received his colleagues' praise as a team-builder and as an expert in areas such as foreign relations, environmental sustainability and National Defence.
Among the positions he held as MP were Parliamentary Secretary to the Minister of Finance, Junior Minister of Environment and Critic for Foreign Affairs and National Defence.
On the Asia-Pacific file, Bryon has complimented his previous academic expertise with an in-depth perspective gained through his experience planning, organizing and leading successful international trade missions in Asia-Pacific countries; through chairing and participating in a wide range of international parliamentary delegations; and through serving as a Canadian spokesperson at numerous international conferences. In fact, Bryon received the Order of the Rising Sun – Gold/Silver Star from His Imperial Majesty and the Prime Minister of Japan for his tireless work on Canada/Japan relations. He also holds the position of Honorary Consul General for Myanmar.
In the realm of Canadian municipal/provincial relations, Bryon offers expertise and critical relationships that he developed as past President of both the Federation of Canadian Municipalities (FCM) and the Canadian Parks/Recreation Association (CPRA).
Bryon has written extensively on national and international issues. He holds a M.A., B.A. and B.Ed. from the University of Toronto, as well as a diploma in municipal administration – Accredited Municipal/Clerk Treasurer (AMCT) from St. Lawrence College. He also holds an Institute of Corporate Directors Designation (ICD.D) from the Rotman School – University of Toronto.
- Tel: 888-439-8988 Ext 4
- Location: TORONTO
- Email: firstname.lastname@example.com


text:
Another set of unique die cut labels, exclusive to our studio - you won't find them anywhere else. They are so versatile, you will want to stick them everywhere! You can also have custom wording added, in case you want these labels to seal up party favors, or perhaps for product packaging. You choose what text goes on the labels, we format & print it for you - and you get sheets of cute labels to use anywhere.
Shape ONE is 2.4" wide x 1.5" highShape TWO is 2.25" wide x 1.7" high.
These labels are sold in sets of 36, with 12 labels on each sheet - you will therefore receive three sheets of labels.
Any custom wording will be printed the same on all labels. Individual text can not be placed on the labels unless you contact us ahead of time for an estimate. Thank you.
Adding product to your cart


text: Everything is a Remix, Part II is out today, and for me it's not a moment too soon. As I wade further into this project of shameless solipsism and word-wasting - which will likely be almost entirely derivative of other people's thoughts and observations - it's good to be reminded that there's no such thing on earth as "pure originality." If I can add just a little to each thing I steal, then maybe it's worth it. How little counts as "just a little"? We'll see.


text:
Ending breast cancer forever is our mission, a goal we strive toward each and every day. We know we won't achieve it alone—that's why we've long believed in the power of partnerships. We collaborate with the most impactful people and organizations around the world to help leverage every dollar invested, expand our scope and capabilities and extend the benefits of our work. And we do it to ensure that we can make the biggest impact for women and men with breast cancer.
In addition to the more than 2,500 research grantees and 1,500 Affiliate partner organizations, Komen also works with over 60 national and international partner organizations and alliances to support:
Helen Keller
Learn how we're making a measurable difference through alliances and community partnerships.
Discover the resources resulting from partnerships that help accelerate the progress of our researchers and supporters.
Read about the scientific and patient-centered conferences we support to encourage the exchange of information and the empowerment of patients.


text:
As we get close to the November elections, it is important to remember that there are some politicians who have the courage and principles to speak truth to power. Mitt Romney's father, George Romney, was one of them.
George Wilcken Romney (July 8, 1907 – July 26, 1995) was an American businessman and Republican Party politician. He was chairman and president of the American Motors Corporation from 1954 to 1962, the 43rd Governor of Michigan from 1963 to 1969, and the United States Secretary of Housing and Urban Development from 1969 to 1973.
Romney ran for Governor of Michigan in 1962 as an independent-minded reformer defending the individual against the power of "Big Labor, Big Industry, and Big Government." He was elected and then subsequently re-elected in 1964 and 1966 with increasingly large support. He succeeded in attracting businesses to the state and in cutting unemployment to below the national average. Romney had also inherited a substantial budget deficit, but he left office with a surplus. Romney also led the way for a large increase in state spending on education, and Michigan thereby began to develop one of the nation's most comprehensive systems of higher education. (I went to the University of Michigan — Go Blue.)
In the 1964 U.S. presidential election, Senator Barry Goldwater quickly became the likely Republican Party nominee. Goldwater represented a new wave of American conservatism, of which the moderate Romney was not a part. Romney declared, "If [Goldwater's] views deviate as indicated from the heritage of our Party, I will do everything within my power to keep him from becoming the Party's Presidential nominee." During the Fall 1964 general election, Romney cut himself off from the national ticket, refusing to appear on the same stage with Goldwater. When pressed at campaign appearances about whether he was supporting Goldwater, he replied, "You know darn well I'm not!" Romney was re-elected as Governor of Michigan in 1964 by a large margin, despite Goldwater's landslide defeat to President Lyndon B. Johnson that swept away many other Republican candidates.
We need more politicians like George Romney in both parties, and we need them to work with each other to produce the kinds of results that George Romney produced in Michigan.
Click here to read more about George Romney and other historic Liberal Republicans.


text:
I don't know about you but Monday mornings aren't something I look forward to. To be honest I'm always very very sleepy. It doesn't matter how early I go to bed the night before… Mondays are always Mondays.
Well, to make things easier I try to think about what I'm going to wear the night before. This way I won't be rushing in the morning (as I normally do).
I picked up this very simple outfit for today. Perfect for those who work in an office, and also appropriate for going for a drink after work, if the opportunity comes up.
The shirt is from Zara Woman. I bought it when I went to Spain for Christmas but they also had it in the UK online shop.
Black jeans – the ones in the pic are from Mango.
I found the belt in a leather shop in Camden Town Market stables.
I'm wearing it with black ankle boots but I think it would also look nice with a pair of stilettos, if you are feeling bold.
What is your Monday outfit?