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Medicial Mistakes?
How many people each year suffer some type of preventable harm that contributes to their death after a hospital visit?
from 46,000 to 78,000
from 78,000 to 132,000
from 132,000 to 210,000
from 210,000 to 440,000
Interviews with People Who Make a Difference : The Evolving Cayce Legacy
Interview with Charles Thomas Cayce
as interviewed by Daniel Redwood DC
Dr. Charles Thomas Cayce, a soft-spoken, sensitive, and thoughtful man in his early fifties, is keeping alive a family legacy spanning back three generations to his grandfather, Edgar Cayce (1877-1945), the best-known and most widely-respected psychic of modern times.
A child psychologist who has done extensive work with psychic young people, Cayce is the President of the Edgar Cayce Foundation, and serves on the executive board of the Association for Research and Enlightenment. Both organizations are headquartered in Virginia Beach, Virginia.
Both in Virginia Beach and in outreach programs throughout the United States and much of the western world, the A.R.E. sponsors programs in such areas as holistic health, meditation, dreams, reincarnation, and spiritual healing. Central to its mission is the dissemination of the material in the more than 14,000 psychic readings given by Edgar Cayce. The A.R.E. Library, where the readings are preserved and catalogued, contains what is reputed to be the largest collection of metaphysical and holistic health works in existence.
In this interview with Dr. Daniel Redwood, Dr. Cayce speaks with characteristic presence of mind and absence of pretension. In addition to answering questions about his grandfather's work, he tells the touching, personal story of his first experience with meditation, as a young child cradled in his father's arms in the wee hours of the night. His comments on the Edgar Cayce predictions of earth changes are also of exceptional interest.
For you, what is the central message in Edgar Cayce's work?
For most people who come in contact with the work of my grandfather, the central message is
that there is more to us than the physical body.
Edgar Cayce for the most part gave individual readings for people with specific problems. He was not so much responding to general questions with discourses on how to treat this physical illness or that kind of interpersonal relationship, but responding to specific individual questions.
To a large extent, the people who came to him with those questions were wrestling with that basic issue. Their physical body was hurting, or they had just had a death in the immediate family, or a divorce, or some other of the sorts of crises that are age-old, and typical in our culture. The message in the Cayce readings is: "There is more than this." More than your heart problem (that is, your physical heart problem), more than this or that. He talked about this in many different ways, and made suggestions about how to better come to know the "more," call it spirit or soul or whatever.
Thousands of the Cayce readings dealt with information intended to aid in healing the sick. The holistic emphasis on body, mind, and spirit as an indivisible unity was highly unusual for its time, and set the stage for so much that came later. When the AMA Journal had an editorial on holistic health several years ago, it dated the movement's inception to the early years of Edgar Cayce. How is the A.R.E. preserving and developing this healing legacy today, particularly through the A.R.E. Clinic in Arizona, and the Reilly School of Massotherapy in Virginia Beach?
The clinic in Phoenix was established by two medical doctors, a husband and wife, Gladys and Bill McGarey. They had gotten very interested in trying to apply some of the treatments and suggestions from the Cayce readings in their medical practice. They had six children and an ongoing practice in Phoenix, and weren't ready to move to Virginia Beach, so they worked with my father, Hugh Lynn Cayce, to set up a clinic in Arizona to work with and test the ideas in the Cayce readings. That has now been going for about 20 years, and has grown from a staff of two to a staff of seventy, with all sorts of health care professionals involved now: massotherapists, chiropractors, osteopaths, and biofeedback technicians, nurses, medical doctors, and many others. They have broadened their area of interest beyond the Cayce readings, to acupuncture and the whole field of holistic medicine.
In fact, the A.R.E. Clinic and the health care professionals who were connected with the clinic, either through conferences held by the clinic or as members of the staff, to a large extent were responsible for helping to establish the American Holistic Medical Association. A more recent focus in working with the health information in the readings, is the establishment of the school of massotherapy here in Virginia Beach, which has a 600-hour curriculum. In addition to learning massage, there are courses on nutrition, meditation, dreams, and of course anatomy, physiology, and so forth. There is a wide spectrum covered. I often think of the school of massotherapy as a school of healing, or a school around which a philosophy of healing is built and taught. The Cayce readings are a central part of that.
Edgar Cayce's most famous readings are probably those dealing with holistic health, reincarnation, and earth changes. Many of the conferences in Virginia Beach focus on these topics. The ongoing A.R.E. study groups, on the other hand, seem to deal more with the moral and spiritual dimensions of day-to-day living. How important are these study groups to the A.R.E.?
They are at the heart of what the A.R.E. is about. The study groups started in the early 1930s, when a group of people who were working with Edgar Cayce, living nearby and meeting with him pretty much every day as he was giving readings, decided as a group to get some readings and try to work together on their psychic development. Out of this came the Search For God study groups.
The readings that Edgar Cayce gave on this subject of psychic development had as a focus the idea that psychic development needs to come as a kind of side effect of spiritual growth. The focus should not be so much on learning how to see auras, or learning this or that psychic ability, but on the spiritual. As that happens, the psychic sensitivity will be enhanced. Two books,
A Search For God, Volumes One and Two,
each containing twelve chapters, form a sequence of steps for discussion and application for spiritual growth. The first chapter in the first book is not "How To See Auras." Instead, it's "Meditation."
There are a couple of thousand study groups meeting weekly around the country. It's not a class with a teacher or a leader. It's more a discussion group format, with everyone providing input based on their personal experiences.
A major important element in the Search For God groups, is the decision by group members to work with a particular tool or idea in the upcoming week, and to come back the following week and talk about how we did. That moves the group from a discussion group to one of application. There is a continual testing and applying of the ideas that are being discussed. The most exciting discussions I've had with people about what they have found to be helpful in the Cayce readings, have come with people who are plugged into these regular Search For God meetings.
Many people have understood the Cayce readings to predict some fairly apocalyptic earth changes in the relatively near future, including the destruction of much of California and the submerging of many coastal lands, where so much of the world's population is centered. How accurate have the Cayce readings been thus far with regard to earth changes, and how seriously should people take these warnings? Is any of it inevitable?
The Cayce readings were, with a few exceptions, not very specific in terms of dates for earth changes. There were certainly some specific geographic areas talked about -p; Japan and Southern California among them. The dates were not given in a very specific way, although there was a period between 1958 and 1998 talked about often, as a time of accelerated change, a time when there would be changes in many aspects of our culture. Many people have interpreted the Cayce readings to suggest that these earth changes would occur between '58 and '98, particularly in the latter half of the 40-year period.
One specific date and location that's often quoted is the prediction that evidence of Atlantis would be discovered as a result of land rising off the coast of Florida, near Bimini, around '68 or '69. This reading was given in the '30s or early '40s. Many people interpreted this to mean that land would arise and the lost continent and civilization of Atlantis would be discovered, that it would pop up from the ocean at that time. That really didn't happen, as far as anyone can tell, unless it popped up and then popped back down.
So either that was a mistake, an inaccuracy with regard to earth changes in the Cayce readings, or there is some other interpretation of "land rising." In any event, every time earth changes were spoken about in the Cayce readings, it was almost like an Old Testament prophecy, in the sense of admonishing us that our thoughts can and do influence physical matter.
It makes a difference what we think and how we act.
It makes a difference not only in the mental realm, but in the physical realm.
The readings were saying, way back in the '20s and '30s, that if we don't get our act together, the influence can be such as to even produce earth changes. The readings would go on to say that just as our thoughts can produce changes in physical matter, they can also prevent those things from happening. So the Cayce readings clearly do not suggest a kind of fatalistic approach to earth changes. They suggest that with a more constructive attitude toward our planet and toward each other with our thoughts, earth changes will not be necessary.
With regard to the '58 to '98 period, there is another part of the Cayce readings that suggests the Second Coming at the end of this age. There are references in the Cayce readings to a "New Age" beginning around this time, and of course that term is being tossed around lots these days.
So there are two scenarios in the Cayce readings; one an apocalyptic scenario of major destruction as a result of earth changes or perhaps misuse of technology, and the other a New Age in the most positive and perhaps esoteric sense -p; a Second Coming of the Christ or some other blossoming of spiritual reawakening that will manifest in a very profound way.
The Cayce readings address both of these possibilities, stating essentially that it can go either way. But the constant in the answers to both those sorts of questions is that for you or for me, and I guess this can be misinterpreted, for you and me
it doesn't really matter.
Whatever the future may bring, our job is to become more spiritually attuned, so that we can hear spirit speak clearly to us as to what we should be doing, regardless of what happens. We are called upon to get our act together as individuals with regard to how we are going to help our fellow man. Those two basic steps, of attunement and service, are crucial.
I have seen certain individuals whose psychic abilities were remarkable. I have seen others who present themselves as psychics and have been unimpressive. How does one distinguish a truly gifted psychic from a charlatan, or someone who is sincere but deluding himself?
There are several helpful measuring sticks, which need to be used in combination. We should take an approach similar to what we might do when deciding to visit a chiropractor, a dentist, or some other health care professional. We get recommendations from people we trust. I don't think it's usually the best strategy to simply flip open the Yellow Pages and throw a dart at the thing, you know? Or to go by who happens to be most available, or in closest proximity. We need to get positive recommendations from people we are on the path with.
We also need to evaluate whether the person who is professing to have psychic abilities is following their own advice. If they are talking about changes in diet, and meditating regularly, and they are not doing that in their own life, then a major question mark is raised, at least for me.
It is also important that the information the person is purportedly giving, must point beyond itself. At a gut level, it needs to awaken something inside us that points to something higher. What chakra feels stimulated when we are with the person, or when we hear them speak? If it's not one of the higher centers, then I think questions should be raised. Not that people with psychic ability are going to score 100% on all of these criteria, but these are the sorts of questions we need to ask ourselves when trying to evaluate people with psychic ability.
Speaking as psychologist and as a Cayce, how healthy would you say it is to base one's actions entirely on the advice of a psychic?
I would never do that. The Cayce readings are really clear about this. There is great emphasis placed on our responsibility for what I call "doing our homework." The readings that Edgar Cayce gave rarely, if ever, made decisions for people. People tried to ask for decisions: "Should I get a divorce?" or "Should I change to this job?" But virtually never did the Cayce readings make those decisions for people.
The readings suggested that our purpose on the earth is to learn to make our will one with God's will. This is a simple little phrase, but very complex when we try to apply it. I think if we forego the opportunity to make decisions, and try to get psychics to do that for us, then we are foregoing opportunities to grow spiritually on the earth, forgoing our major purpose for being in the earth.
So I would see using sources of psychic information, whether it's our own, in terms of dreams or insights as a result of meditation, or whether it's from someone else, as an adjunct to our own mental faculties and our own choice-making faculties.
How can we best nurture the intuitive capacities in ourselves and our children?
Each of your questions is worthy of a day-long answer. I hope I'm picking the high points when I address them. With regard to children, that question was asked over and over in the Cayce readings. There were parents whose children may have been having psychic experiences, or who themselves had been helped by the readings and were interested in sharing those sorts of concepts with their children. They would ask, "How should we do this?"
The most frequent answer was, to paraphrase it in more contemporary language, "Don't lay a heavy trip on the children. Teach by example rather than precept." I remember growing up in a family where this kind of thing was talked about all the time. I assumed that everyone talked this way. We would talk about dreams at the breakfast table. There was a meditation time every day. People would always be visiting our home, talking about reincarnation and psychic experiences. I heard my father talk about meditation from the time I remember my parents talking about anything. I came to lectures at the A.R.E. as a child of 6, 7, or 8, about meditation and similar subjects. I don't even remember my parents pushing me about starting to meditate myself, or putting me in classes to learn to meditate, or anything like that.
My father was meditating at 2 in the morning for an extended period when I was a child. We had just moved to a different house. I guess I was 7 or 8 years old. He would get up and come by the kids' rooms, down the hall from their bedroom, and use this unfinished bathroom that was like a closet. He would close the door, and use that area to meditate. Sometimes I would wake up when he would come down the hall and I would go to the door and listen. Several times I would call to him and ask what he was doing. He would say something about meditation, but I couldn't understand what he was saying.
One day, one morning, I woke up and he was in the room meditating. I stood by the door, and I was fascinated. I couldn't hear anything in the room, and I kept wondering what he was doing, why he would do that every day. Anyway, on that particular morning I sat down outside the door to wait, and I fell asleep. He finished meditating, and opened the door, and I kind of fell over into the room. He picked me up and carried me out into the hall. He sat down on the floor, and it was dark. It was a linoleum floor, and I remember it feeling cold on my feet. He sat cross-legged against the wall, and kind of tucked me into his lap, and said something like, "Well, let's just sit here a minute and see what this meditation thing is about."
The next thing I remember is that I saw light. My chin was against my chest, and rather than raise my head, I just rolled my eyes up to look at the ceiling lights, to see if the lights were on. I was kind of half asleep and didn't feel like raising my head. I looked up, and the lights were not on. I felt very warm and tucked in, and that's the last thing I remember before waking up in bed the next morning. I remember lying there in bed that morning thinking that I now knew what meditation was.
I later asked my father how he did that. I wanted him to do it again! [Laughter]. He said he didn't do anything special. He said that what I needed to remember, as a possible future parent, was that parents must be sensitive to times when children are particularly open to an experience. It may be in the middle of the night when their brother or sister is sick, or at some other very inopportune time for the parent.
There is no question that it would have been easier for my father to go back to bed, to pick me up and put me to bed when I rolled in the room. He had to go to work the next day. But he sensed that here was a chance to be with me and experience meditation. And that meant so much more than all the words he had tried to say to me about meditation. I think that if they had pushed with any of this stuff as I was growing up I ran from it for a long time, and I think I would still be running if they had pushed. Parents need to be very careful. Many of us, whether we're parents or whatever role we're playing, as we get involved with these sorts of ideas, often we get very excited and enthusiastic and we tend to want to beat other people over the head, sort of force-feed them, proselytizing. That doesn't work, and it especially doesn't work for children.
To preach at them and push, especially if we are not practicing these things ourselves, just causes more problems.
I have heard that you and your family live on a farm with sheep and no television. Does city life leave something to be desired?
The short answer is yes. In my opinion, city life does leave something to be desired. The no-TV is primarily the work, the instigation, of my wife. I don't want to knock TV, but we are concerned about both the content of the programs and the energy involved in children planting themselves in front of a television for inordinate amounts of time.
We both work. My wife works part-time as a social worker at a hospital in Norfolk, and we don't have a lot of time together as a family. To use up that time with any of us, or all of us, sitting in front of this little box looking at pictures rather than having other experiences that are for us more filled with life and love, is kind of a waste.
With the farm, for me it is a great counterbalance to spending lots of time with people. To be able to dig in the dirt, or milk a cow as I used to. We have a real menagerie with all sorts of animals and birds. To interact with them provides a balance. I think a farm is also a wonderful place for children to grow up and experience all of the major cycles of life -p; birth and death and the bonding between mothers and infants. It's a great environment for children to get a healthy taste of life and death.
We are really committed to growing our own food, trying to work with our diet in that way. The farm environment fulfills many needs for us. I feel really lucky to be able to be in that situation.
What is your fondest dream?
Let me relate that to the A.R.E., my fondest dream for the A.R.E. That would be for the A.R.E. to go out of business. I really mean that. That has problems connected with it, because I certainly feel a responsibility for the people who are working here and all of that. But I think the role of the A.R.E., through my grandfather's readings, is to help people awaken to their spiritual nature, to help people see ways to manifest that spiritual nature in this physical world. I think there are lots of structures around that are trying to help us do that. Certainly that's a mission of the church. It would just be wonderful if that were to happen more and more, to the point where those structures weren't necessary, including the A.R.E. So it's certainly idealistic, but that would be very exciting.
To have succeeded to the point where the institution was no longer necessary.
I have one more question. What major new endeavors is the A.R.E. involved with at this time?
One is a Master's Degree in Transpersonal Studies, which was just certified by the State of Virginia. We are now offering this degree under the auspices of Atlantic University, which started here as a full-time university back in the early '30s and now exists as simply this one master's level program. I am particularly excited about it because I think it will afford more of an opportunity for both faculty and students to do research on ideas in the Cayce readings, particularly from a comparative perspective. Then we can make that research available, through articles in our magazine or in other journals. The master's theses that will be done, the preparation by the faculty for the courses they'll be teaching, will provide a major additional angle of research on the ideas in the Cayce readings.
I am also, from a research standpoint, excited about the process of putting the readings and all of the corollary information on computer. The software allows for things like key-word searches, so that you in a matter of seconds can have the computer scan the entire body of 14,000 reading for the information you're seeking. This is fantastic for health care professionals, other researchers, and interested A.R.E. members.
Daniel Redwood is a chiropractor and writer who lives in Virginia Beach, Virginia. He is the author of A Time to Heal: How to Reap the Benefits of Holistic Health (A.R.E. Press), and is a member of the editorial board of the Journal of Alternative and Complementary Medicine. He can be reached by e-mail at email@example.com.
©1990, 1995 Daniel Redwood
See all interviews by Daniel Redwood DC
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About The Author
Daniel Redwood, DC, is a Professor at Cleveland Chiropractic College - Kansas City. He is editor-in-chief of Health Insights Today (www.healthinsightstoday.com) and serves on the editorial boards of the Journal of the...
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via press note:
Last night's late starting post Super Bowl broadcast of Elementary (11:15PM-12:15AM) averaged 12.0/24 in the metered markets and is on track to being the freshman drama's most watched episode and highest rated in key demographics. The 11:15 start is the latest for any post Super Bowl program
Late Late Show with Craig Ferguson (12:45-1:45 AM) averaged a preliminary 3.2/11.
Time zone adjusted fast nationals will be available later today.

subset: commoncrawl


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10 Easy Jams & Pickles for Beginners
We have reached the tipping point of summer when we stop longing for our favorite fruits and vegetables to finally arrive and start wondering what the heck we're going to do with it all. Those of you with CSAs definitely know what I'm talking about! If you're new to the canning and preserving party, never fear: We have ten fantastic recipes that teach you the ropes — and help you take care of all that fresh produce.
Small-Batch Garlic Dill Pickles
If you have never canned a thing in your life, the best place to start is with an easy refrigerator jam or a batch of quick-pickles — like our peach and plum jam, or pickled radishes. Neither of these involves actual canning (phew!), but they're a great way to both make something tasty with your produce and to get a feel for transforming fresh fruits and vegetables into preserved foods.
When you're ready for the next step and feel up for some actual canning, try making a small batch of Garlic Dill Pickles — just three pints! Again, this is a good way to ease into the process of canning and pickling without it taking over your kitchen. Want some jam? Our 3-Day Apricot Jam might seem intimidating at first, but it's actually all about breaking the long jamming process down into bite-sized chunks.
Jams and pickles — you can totally do it! Grab yourself some cucumbers and a pint of berries, and get to it.
What are you canning and preserving right now?
- 1 Pickled Yellow Squash
- 2 Small-Batch Garlic Dill Pickles
- 3 Peach, Plum, and Ginger Jam
- 4 Garlic & Coriander Quick-Pickled Baby Carrots
- 5 Strawberry Refrigerator Jam
- 6 Pretty-In-Pink Pickled Radishes
- 7 Quick-Pickled Berries
- 8 3-Day Apricot Jam
- 9 Easy Japanese Pickled Cucumbers
- 10 Plum & Earl Grey Preserves
(Images: See linked recipes for full image credits)


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Nature. Vol. 345. 7 June, 1990
Click Here to See Cover Photo
David Vaux*, John Tooze & Stephen Fuller ^ #
* Cell Biology and ^ Biological Structures Programmes,
European Molecular Biology Laboratory,
Meyerhofstrasse 1, 6900 Heidelberg, FRG
# To whom correspondence should be addressed.
Monoclonal antibodies were raised against antibodies to distinct carboxy terminal KDEL sequences of two soluble, resident endoplasmic reticulum proteins. These anti-idiotype reagents recognize an intrinsic membrane protein with characteristics expected of a receptor responsible for the recognition and return of resident proteins to the endoplasmic reticulum.
EUKARYOTIC secretory proteins begin their journey out of the cell by co-translational translocation into the lumen of the endoplasmic reticulum (ER)1. Vesicular transport 2 of these soluble proteins to the Golgi apparatus seems to be by a nonspecific fluid phase transport that does not require a specific signal 3. The presence of several resident soluble proteins in the lumen of the ER provides an apparent exception to this hypothesis of nonspecific fluid phase transport 4. Several of these resident proteins mediate the proper assembly and folding of newly synthesized proteins. They include the immunoglobulin heavy chain-binding protein (BiP), which apparently functions in protein oligomerization 5, and protein disulphide isomerase (PDI), which catalyses the rearrangement of disulphide bonds 6. These retained soluble ER proteins are not a minor population; from data in the literature 7,8 we calculate that PDI is a very abundant protein of the ER, present at a concentration of >400 µM.
Peptides were synthesized in an automated continuous flow instrument using FMOC chemistry 29 and purified by reverse phase HPLC. The peptide KXXXXXKDEL consists of a mixture of analogues containing any one of alanine, aspartic acid, histidine, glutamine, leucine, tyrosine or lysine at each of the positions X. KDEL-terminated peptides are identified throughout by their unique amino-terminal residues. Single-letter amino-acid code used.
A clue to the mechanism of retention in the ER came from the finding that BiP and PDI share a carboxy-terminal sequence (Table 1), Lys-Asp-Glu-Leu (KDEL) with other resident ER proteins including glucose-regulated protein 94 (GRP 94) (ref. 9). In addition, a secreted soluble protein, lysozyme, can be converted into a retained protein of the ER by adding the last six amino acids of BiP, Ser-Glu-Lys-Asp-Glu-Leu, to the carboxy terminus. Addition of a further two amino acids (GlyLeu; GL) to the carboxy terminus results in secretion of the mutant Iysozyme, as do numerous other changes in the KDEL terminus 10. Thus, the presence of KDEL at the carboxy terminus is sufficient for the retention of a protein in the ER. A similar retention system functions, albeit less efficiently, in the yeast Saccharomyces cerevisiae 11, although a different recognition signal, His-Asp-Glu-Leu (HDEL), is used.
The retention of resident proteins bearing the KDEL signal by interaction with a static receptor within the ER seems unlikely: the proteins that must be retained by this receptor are far more abundant than any single candidate receptor polypeptide. It has been suggested that the receptor might function in a distal compartment by recognizing the KDEL signal on escaping resident proteins and mediating their return to the ER 10. Microinjection experiments 12 demonstrate that the presence of a KDEL tail on BiP does not significantly alter its rate of diffusion in the ER from that of BiP bearing the unrecognized KDELGL tail. Both diffuse more rapidly than the integral membrane protein, haemagglutinin, but more slowly than the secreted protein, albumin. Hence, the KDEL signal is not continually and tightly bound to a component of the ER membrane although proteins bearing this signal probably interact weakly within the ER. Proteins with the KDEL signal do leave the ER transiently, as shown by the modification of a cathepsin D hybrid protein bearing the KDEL tail 13. Thus, resident ER proteins presumably pass through a distal compartment that is proximal to the classical Golgi apparatus before returning to the ER 4. Compartments have been identified between the ER and classical Golgi by a variety of approaches 14-16. Although the relationship between these structures remains unclear, we will refer to them collectively as the salvage compartment 4.
The identification of a receptor for the KDEL signal must rest on several assumptions about its properties. First, we assumed that all KDEL signals must be recognized by a single receptor species that does not bind unrecognized sequences such as K . . . GL. Second, we assumed that the receptor is a recycling protein and must have two distinct binding states. While functioning, the receptor must alternate between a high-affinity state that recognizes escaped KDEL-bearing proteins and a low affinity state that releases them into the lumen of the ER. Conventional ligand affinity methods for receptor identification are problematic because the ionic environment of the ER and salvage compartment are not well defined. We made broad assumptions about receptor localization. The receptor could be present in the ER, in transitional elements, in the salvage compartment, in proximal parts of the Golgi apparatus and in transport vesicles between these compartments. For these reasons we used an immunological approach to search for a protein with the properties required of the receptor.
Monoclonal internal image anti-idiotype antibodies are powerful tools for the identification and characterization of the participants in a receptor-ligand interaction 17-18. Peptides containing the KDEL signal (Table 1) can be used to raise anti-KDEL antibodies that recognize this signal at the carboxy terminus of ligands. Internal image anti-idiotype antibodies 18-20 raised against these anti-KDEL antibodies should then recognize the receptor by its KDEL-binding site. We raised such anti-idiotype antibodies and demonstrate here that they recognize an intrinsic membrane protein that is a candidate for the KDEL receptor.
Antibody generation
Synthetic peptides corresponding to a variety of carboxy-terminal sequences (Table 1) were used to generate polyclonal and monoclonal antibodies. Rabbit antisera to some of these peptides have already been shown to recognize the corresponding ER proteins and to label the ER by electron microscope (EM) immunocytochemistry 21; a more complete characterization of these antibodies will be presented elsewhere (S.F., J.T. and D.V., manuscript in preparation). An important assumption of the anti-idiotype approach is that the first round antibodies recognize the KDEL retention signal itself on resident ER proteins. As the signal specificity lies within the last four residues, recognition by our first round antibodies must be dependent on the carboxy terminus. A monoclonal anti-PDI tail antibody was generated by immunization with peptide KDDD (Table 1). This IgG antibody (1D3) recognizes a double band of relative molecular mass ~55,000 (Mr~ 55K) in western blots, consisting of PDI and calreticulin 22, labels the ER by EM immunocytochemistry 21 and gives a clear reticular pattern by immunofluorescence (Fig. 7h). Figure 1 confirms that the signal recognized by 1D3 can be removed from PDI by gentle incubation with carboxypeptidase A under conditions that do not change the apparent protein pattern of the blotted lysate and do not affect the recognition of PDI by a polyclonal antiserum raised against rat PDI. Thus, 1D3 recognition depends on carboxy-terminal residues. Taken in conjunction with solid phase assays on a range of synthetic peptides (data not shown), this result leads us to conclude that 1D3 recognizes the retention signal in PDI and calreticulin. Similarly, polyclonal antisera to the KETE peptide (Table I) recognize specifically the carboxy-terminal residues of BiP, PDI, GRP 94 and other proteins of the ER (S.F., J.T. and D.V., manuscript in preparation).
FIG. 1 Monoclonal antibody, 1D3, recognizes
a carboxy-terminal determinant on an ER protein, PDI. Western
blot using monoclonal antibody 1D3 (lanes A, B, C) or rabbit anti-PDI
antiserum (lanes D, E) on total cellular protein before (lanes
C, E) and after treatment with carboxypeptidase A at low concentration
(lane A) or at high concentration (lanes B, D). The arrow identifies
METHODS. VG mouse hybridoma total cell protein was separated by gel electrophoresis30 and electrophoretically transferred to nitrocellulose using a Genie electroblotter (Idea Scientific, Inc.) according to the manufacturers instructions. Strips from a single blot were washed in 2% (v/v) Tween20 in 25 mM Tris-HCl buffer 150 mM NaCl (TN), pH 7.6, and incubated overnight at 37°C in 25 ml of 2% Tween-20 TN pH 5.5 buffer, other incubations contained a 1:250 or a 1:2500 dilution of carboxypeptidase A (Sigma). The filters were then washed in 2% Tween-20 TN pH 7.6, blocked with BLOTTO31, and probed using either monoclonal antibody 1D3 (prepared against KDDD peptide coupled to Keyhole Limpet Hemocyanin (KLH)) or a rabbit antiserum to PDI (rabbit immunized as described 21 with protein purified by the method of Lambert and Freedman 32) followed by appropriate affinity purified alkaline phosphatase-conjugated goat second antibodies 33. Two-dimensional gel electrophoresis followed by western blotting with anti-KEEE antiserum confirmed that the second band recognized by 1D3 is calreticulin (lane C).
Two approaches were used to prepare anti-idiotype antibodies to the anti-tail antibodies. First, monoclonal anti-idiotype antibodies were generated by the method of paired in vitro immunization 18 using unconjugated KETE peptide as the starting immunogen. The second-round hybridomas were screened for ER- or Golgi-related staining patterns by indirect immunofluorescence. Two wells were selected for further characterization, both of which yielded stable IgM-secreting hybridoma cell lines after cloning in agarose. The results obtained with the line designated 5D3 are described below. Second, syngeneic mice were immunized with fixed cells of the 1D3 hybridoma cell line. Hybridomas resulting from this in vivo immunization were screened for reactivity with anti-KDEL antibodies (rabbit anti-KAVK). Of 256 hybrid-containing wells, 15 gave a strong reaction. The results for a stable, cloned IgG-secreting line from one of these, 9D6, are described below.
Anti-anti-KDEL antibodies
Immunoblotting of lysates of control murine hybridoma cells (VG cells) revealed a major immunoreactive band of 72K with 5D3 and 9D6 (Fig. 2a). This 72K antigen remained with the membrane pellet after carbonate-washing of microsomes up to pH 11 (Fig. 2e). The 72K antigen also partitioned into the detergent phase on separation of a TX-114 lysate of VG cells (not shown). Thus, the antigen is presumably an integral membrane protein 23. Protease protection experiments revealed that a 54K immunoreactive fragment remained resistant to protease treatment of intact VG cell microsomes (Fig. 2c). Neither band was present when protease treatment followed detergent lysis, and blotting with anti-PDI showed that this luminal ER protein was completely protected under the conditions of Fig. 2c, lane 2 (not shown). Immunoblotting with 5D3 showed that the antigen was present in all the vertebrate species tested but not in S. cerevisiae (data not shown).
FIG. 2 Both monoclonal anti-idiotype antibodies
recognize the same integral membrane protein. The total post-nuclear
lysate from the mouse hybridoma cell line VG was blotted with
the monoclonal antibodies 5D3 (a, lane 1) and 9D6 (a,
lane 2). Mr (in K) marker positions for a are
indicated to the left. Antigen was immuno-precipitated from [35S]methionine-labelled
9D6 (b, lane 1) or 5D3 (b, lane 2) cell lysates. The cross
reactivity of the 5D3 and 9D6 antigens is demonstrated by the
isolation of the immunoglobulin-antigen complex from supernatant
of 9D6 hybridomas with fixed S. aureus cells and immunoblotting
with 5D3 (b, lane 3). VG microsomes treated with proteinase K
and blotted with 5D3 show that a cytoplasmic portion of the antigen
is accessible to cleavage and that the antigenic site is contained
within the protected lumenal domain (c, lane 2, solid arrow;
undigested antigen, c, lane 1 open arrow). Blotting of
an equal amount of protein from total post-nuclear lysates from
5D3 (d, lane 1), 9D6 (d, lane 2) or VG (d,
lane 3) with the monoclonal antibody 5D3 shows the increased level
of antigen (solid arrows) expression in the anti-idiotype hybridomas.
VG lysate was immunoblotted with 1D3 ascites using a µ-chain
specific antibody to show IgM reactivity (d, lane 5), which
was absent when 1D3 supernatant was used (d, lane 4). VG
lysate was also western blotted with polyclonal mouse antisera
raised against the isolated 5D3 antigen (d lane 6). The position
of the mouse heavy chain in VG cell lysates is marked by the open
arrow. e, Demonstration of the membrane-associated nature
of the 5D3/9D6 antigen by carbonate washing of VG microsomes at
various pH (lane 1, pH 11.6; 2, pH 11.0; 3, pH 10.5; 4, pH 10.0;
5, pH 9.5) followed by western blotting of the pellet (lanes 1-5)
or supernatant (lanes 1'-5') with 5D3. The 84K marker (lane M)
and noncarbonate-treated microsomes (lane C) are also shown.
METHODS. Monoclonal antibody 5D3 was produced by paired in vitro immunization 18 using 100 µg unconjugated HPLC-purified KETE peptide as starting antigen. Monoclonal antibody 9D6 was produced by fusing Ag8 myeloma cells to spleen cells from a BALB/c mouse immunized with washed 3% (w/v) paraformaldehyde-fixed 1D3 (anti-PDI tail) hybridoma cells 34. Washed VG cells at 10 7 ml -1 were lysed in lysis buffer (LB; calcium/magnesium-free PBS containing 1% Nonidet P-40, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1% aprotinin (Sigma), 1 mM EDTA and 10 mM sodium azide) and the post-nuclear fraction separated on 10% polyacrylamide gels. Transfer and western blotting was carried out as in Fig. 1 using appropriate affinity-purified isotype specific goat anti-mouse alkaline phosphatase conjugates (Zymed Inc., San Francisco). Microsomes were prepared from VG cells by resuspending a washed cell pellet in 0.25 M sucrose containing 3 mM imidazole, PMSF and aprotinin, 1 mM MgCl2, 10 mM CaCl2 and passing four times through a ball-bearing cell cracker (after W. Balch, Scripps institute, La Jolla, California) followed by pelleting to 1 h at 100,000g in a Beckman TLA-45 rotor. Microsomes were then subjected to carbonate washing 21 or proteinase K digestion. Proteinase K digestion was for 60 min at 25 °C with 250 µg proteinase K ml-1 in 25 mM Tris-HCl buffer (pH 7.5) containing 10 mM CaCl2, 1 mM 1 mM dibucaine, 100 mM NaCl with or without 1% Triton X-100. Digestion was stopped by the addition of 10 mM PMSF, precipitated with an equal volume of trichloroacetic acid and dissolved in reducing sample buffer before electrophoresis and transfer to nitrocellulose. The 9D6 anti-idiotype secreting hybridoma cells were lysed in LB, immuno-precipitated with goat anti-mouse IgG followed by fixed Staph. A (Zysorbin; Zymed Inc.), washed four times in PBS containing an additional 0.3 M sodium chloride and 1% (v/v) Nonidet P-40, then washed once in water and eluted into reducing sample buffer before electrophoresis and transfer to nitrocellulose.
Radio-labelled antigen could be immunoprecipitated from [35S]methionine labelled lysates of 5D3 or 9D6 hybridoma cells (Fig. 2b) or from lysates of isolated microsomes (data not shown) simply by the addition of the appropriate second antibody and fixed Staphylococcus aureus cells. Pulse-chase analysis (data not shown) demonstrated that this complex existed intracellularly before cell lysis. The antigen immunoprecipitated with 5D3 was recognized in immunoblots by 9D6 and the antigen immunoprecipitated with 9D6 was recognized by 5D3 (Fig. 2b).
A third anti-idiotypic response was produced when 1D3 ascites was generated in unirradiated syngeneic mice. The ascites contained an IgM that was reactive with the same 72K band by western blotting (Fig. 2d, lane 5), but which was absent from 1D3 culture supernatant (Fig. 2d, lane 4). Presumably this is because the intact immune system of the tumour-bearing mice responds to the presence of high levels of 1D3 immunoglobulin by producing a primary (IgM) anti-idiotype response, which includes the internal image anti-idiotype specificity capable of recognizing the putative receptor. Taken together, these results indicate that three different routes of anti-idiotype production generate antibodies that recognize the same 72K conserved membrane protein.
The anti-idiotype antibodies recognize a common idiotope in antisera against KDEL peptides (Fig. 3). For example, 5D3 not only recognizes a rabbit antibody raised against the original KETE peptide but also rabbit antibodies raised against other KDEL peptides (Fig. 3a). The complementary assay (Fig. 3b) showed that the rabbit anti-KDEL antisera recognize 9D6 and 5D3 immunoglobulins more strongly than either the monoclonal antibody, 1D3, or another control antibody. No reactivity was seen against rabbit antibodies raised against two irrelevant peptides (Fig. 3a) or preimmune rabbit serum (Fig. 3a and b). This cross-specificity is particularly striking as some of the individual anti-tail antibodies show a much narrower range of reactivity. For example, a rabbit anti-KDDD antiserum that recognizes PDI does not recognize BiP, although rabbit anti-KEED, which recognizes BiP, does not recognize PDI (D.V., J.T. and S.F. manuscript in preparation).
FIG. 3 The monoclonal anti-idiotype antibodies
recognize an idiotope in rabbit anti-KDEL antisera. Upper panel:
5D3 antibody (lane A) specificaliy recognizes rabbit antisera
to KDEL peptides, but not other rabbit antisera. This recognition
is not seen with other IgM monoclonal antibodies (lane B), nor
with second antibody alone (lane C). Lower panel: Rabbit anti-KDEL
antisera recognize both 5D3 and 9D6 immunoglobulin, but do not
react with other murine monoclonal antibodies (H25 and 1D3). This
effect is not seen with rabbit antisera to other antigens.
METHODS. Polyclonal rabbit antisera to the peptides in Table 1 were prepared35 and concentrated by ammonium sulphate precipitation. For the upper panel, aliquots of concentrated rabbit antisera (1 µl) were dried onto nitrocellulose filters which were western blotted with culture supernatant of 5D3, culture supernatant from a control IgM secreting hybridoma, or blocking buffer and processed as described in Fig. 2. For the lower panel, hybridoma culture supernatant from 5D3 or 9D6 or control hybridomas was spotted onto nitrocellulose and probed with rabbit preimmune serum or antisera to KDEL using the same blotting protocol. In each case, the control antisera (Control 1 and Control 2) to irrelevant peptides had titres on their cognate antigens comparable to those of the anti-KDEL antisera on the immunizing peptides.
If the anti-idiotypes mimic the common structure of KDEL tails recognized by the receptor, then antibodies raised against 5D3 or 9D6 immunoglobulins should show the broad KDEL tail specificity of the receptor itself. This would also be proof that 5D3 and 9D6 are internal image anti idiotype antibodies. Evidence for this was obtained from a third round of immunization. Anti-5D3 immunoglobulin sera were generated in BALB/c mice by immunization with fixed 5D3 hybridoma cells. This antisera specifically recognized peptides terminated with KDEL and not those terminated with K...GL (Fig. 4 a). Furthermore, this recognition extended to KDEL peptides other than the original antigen. A comparable primary IgM response was found in the serum or ascites from nonirradiated mice bearing either anti-idiotype hybridoma as an ascitic tumour (Fig. 4b). These results demonstrate that anti-idiotypes elicited by 5D3 or 9D6 recognize the structural feature common to the KDEL tails. Hence, the protein identified by 5D3 and 9D6 should also recognize the KDEL signal on a variety of proteins, which makes it a good candidate for the role of KDEL receptor.
FIG. 4 Third-round immunizations generate anti-KDEL
speatic reactivity. a, Anti-5D3 antisera specifically recognize
KDEL-terminated peptides. b, Spontaneous IgM responses
in mice bearing anti-idiotype hybridoma tumours also show anti-KDEL
METHODS. a, BALB/c mice were immunized four times, intraperitoneally, with paraformaidehyde fixed 5D3 hybridoma cells (~5 x 106 cells per mouse) at intervals of three weeks and bled 10 days after the final immunization. Sera were diluted 1:500 in DELFIA blocking buffer and assayed in triplicate on KDEL and K...GL peptide substrates by a solid-phase time resolved lanthanide fluorescence assay36. Background values obtained with irrelevant mouse antisera were subtracted to give specific binding. The assay was performed three times using sera from three separate immunized animals and a representative set of results is shown. b, Ascites from unirradiated BALB/c mice carrying either 5D3 or 9D6 hybridoma tumours were clarified by ultracentrifugation and used at dilutions of 1:100 in a solid-phase DELFIA assay in triplicate on KDEL, K...GL and HDEL peptides. A highly purified affinity isolated µ-chain-specific goat anti-mouse IgM second antibody conjugated to a Europium chelate was used as detecting antibody36. This antibody has negligible cross-reactivity with mouse IgG. Although both antibodies were tested on all peptides, values below 2,000 Eu counts (photon counts of Europium fluorescence) are not visible in this representation.
Anti-idiotype secreting hybridoma cells
The properties of the 5D3 and 9D6 cells themselves lend further support to the assignment of the 72K antigen as the KDEL receptor. We have already demonstrated that the antigen interacts with the antibodies intracellularly, so it is not surprising that the hybridomas grew poorly, cloned inefficiently and secreted only small amounts of correctly assembled immunoglobulins. Nevertheless, the cells survived and therefore must have compensated for the presence of anti receptor antibody. There are two mechanisms by which antibody interference with the retention of KDEL proteins could be overcome. Either the KDEL ligands could be coordinately upregulated to maintain functional levels in the lumen of the ER in the face of increased leakage, or the receptor itself could be upregulated to titrate the effect of the inhibiting antibody.
Analysis using a fluorescence-activated cell sorter (FACS) revealed that the anti-idiotype hybridomas exhibited the same steady-state levels of PDI, BiP and GRP 94 as hybridomas producing a variety of irrelevant immunoglobulins (shown for PDI in Fig. 5a-c). Pulse-chase analysis confirmed that the anti-idiotype hybridoma cells do not secrete large quantities of these normally retained KDEL proteins (data not shown). These results imply that the anti-idiotype cells did not overcome the retention defect by increased ligand expression. Instead, the hybridoma cells contained greatly elevated levels of the putative receptor. Both 5D3 and 9D6 cells contain the normal 72K membrane-associated protein and large quantities of a heterogenous soluble form (Fig. 2d). The soluble antigen-antibody complex was also found at high levels in media conditioned by the anti-idiotype hybridoma cells (Fig. 2b). The antigen in this material must have passed through the entire Golgi apparatus as a marked Mr shift was observed after neuraminidase treatment (data not shown). It is important to emphasize that soluble receptor is produced only by the two grossly abnormal anti-idiotype-secreting hybridoma cell lines. No other cell lines have been found that secrete material immunoreactive with the anti-idiotype antibodies.
FIG. 5 a, b, c, PDI content
of rapidly growing SP2/0 myeloma cells (a), 5D3 hybridoma
cells (b) and VG hybridoma cells (c) is equal. d,
Expression of the 5D3/9D6 antigen is not rapidly upregulated as
part of the stress response.
METHODS. For a, b and c, cultures of SP2/0 myeloma cells and 5D3 and VG hybridoma cells in rapid growth were washed twice in PBS, fixed in suspension in methanol at -20 °C for 6 min, washed twice more in PBS and preincubated 1 h with PBS containing 10% (v/v) goat serum and 10 mM sodium azide. The fixed, permeabilized, cells were then incubated with 3 rabbit anti PDI antiserum (Fig. 1) for 1 h, washed twice in PBS, reblocked with the goat serum buffer and labelled for 1 h with a rhodamine-conjugated affinity-purified goat anti-rabbit antibody37. The cells were finally washed in PBS and analysed in an EPICS 4 (Coulter Electronics) FACS. Profiles from 50,000 cells, taken from a representative experiment are shown. For d, Vero cells were grown in normal growth medium (lane C), medium containing 50 ng ml-1 tunicamycin (lane T), glucose-free medium (lane L), or medium containing 0.5 µg ml-1 A23187 (lane A) for 4 h. Cells were then collected, lysed, and equal aliquots analysed by SDS-PAGE and western blotted with 9D6 and a rabbit anti-BiP antiserum (rabbit anti-KEED). The western blots were repeated at several loadings of sample to ensure that neither second antibody nor substrate were limiting. The 9D6 immunoblot shown contains five times the protein-loading of the anti-BiP immunoblot.
The poor growth of the anti-idiotype-secreting hybridoma cells suggested that they might be under stress and prompted us to ask whether any of the observed up-regulation of antigen expression could be attributed to a stress response. In particular, as several of the soluble ER resident proteins are induced by glucose starvation, we tested this treatment for its ability to increase expression of the receptor. Figure 5d shows that the putative receptor was not upregulated under conditions that elicit strong induction of known glucose-regulated proteins. Furthermore, the putative receptor was not induced by treatment for up to 4 hours with the glycosylation inhibitor, tunicamycin, or the calcium ionophore, A23187 (Fig. 5d).
Soluble antigen binds to KDEL peptides and PDI
The soluble forms of the putative receptor, isolated from the medium of overexpressing hybridoma cells, were first tested for binding to KDEL-peptide affinity columns. The isolated receptor bound specifically to the peptides containing the KDEL retention signal, but not to K...GL (Fig. 6a). The observed efficiency of binding was low (typically 3-5% of input counts), presumably because this isolated material differs from the 72K transmembrane receptor in normal cells; it is a soluble fragment, glycosylated by passage through the Golgi apparatus and associated with the anti idiotype antibody. Specific binding is obtained with material isolated from either anti-idiotype hybridoma (Fig. 6a) and is divalent cation-dependent and maximal at physiological pH (data not shown). This indicates that divalent cations may play a part in switching the receptor between high- and low-affinity forms. The affinity of the unoccupied binding sites in the isolated antigen for KDEL ligands was estimated by a complementary experiment. This experiment measures the binding of radio-iodinated PDI to isolated antigen attached to Sepharose beads (Fig. 6b). Analysis of these data yields a dissociation constant Kd of 23 µM. This binding can be competed by incubation with KX5 peptide at a concentration of 100 µM, a value similar to that calculated for KDEL tails in the ER lumen.
FIG. 6 Binding of isolated receptor to KDEL
peptides and PDI. a, Radiolabelled soluble receptor isolated from
either anti-idiotype hybridoma cell line binds to several KDEL
peptides, but not to the K . . . GL peptide. b, lodinated PDI
binds specifically to soluble antigen immobilized on Sepharose
METHODS. 5D3 or 9D6 cells were labelled for 14 h with [35S]methionine and the soluble fragment of the receptor isolated from the supernatant by chromatography on concanavalin A-Sepharose (Pharmacia) according to manufacturers instructions, followed by dialysis against TN containing 1 mM dithiothreitol. a, Peptides were crosslinked to CnBr Sepharose (Pharmacia) according to manufacturer's instructions. The peptide-Sepharose particles (50 µl of a l:l v/v slurry) were preblocked by incubation at room temperature in 1 ml blocking buffer (TN 2% Tween-20, 1 mM CaCl2, 1 mM MgCl2, 20% FCS) for 2 h with gentle agitation, then incubated with constant aliquots of the labelled receptor overnight at room temperature. The peptide-Sepharose was then washed three times in blocking buffer without FCS and counted in a liquid scintillation spectrometer. The results shown are the average of four experiments, with each point in triplicate. Error bars indicate the standard error. Background corresponding to beads alone has been subtracted. In a typical experiment, the background value was 650 counts and the highest sample value was 1,800 counts or 3-5% of input counts. All values except K . . . GL and KEED (5D3) are significantly above background (P < 0.05). Gel electrophoresis of a larger quantity of KSEK-Sepharose after incubation with isolated 5D3 receptor showed that the only radio labelled protein bound co migrated with the soluble form of the receptor. b, PDI was isolated from bovine liver by the method of Lambert and Freedman32 as modified in Tooze et al.32, PDI was iodinated using lodobeads (Pierce) according to manufacturer's instructions at a concentration of 5 µg ml-1 and separated from free label by chromatography on Sepharose G-50. The specific activity of the labelled PDi was 1.05 x 107 c.p.m. µg-1 (0.18 atoms iodine per PDI tail) 9D6 soluble antigen was isolated as above and coupled to cyanogen bromide-activated Sepharose 4B according to manufacturer's instructions at a concentration of ~5 mg per ml of beads. For the binding assay, beads were pre blocked by incubation with Blotto containing 1 mM DTT 1 mM CaCl2 and 1 mM MgCl2 for more than 1 h at room temperature and then incubated 1 h at room temperature with 35,000 c.p.m. ml-1 of labelled PDI in the same buffer, together with unlabelled PDI at concentrations varying between 0 and 3 mM. Beads were then pelleted and the percentage of free counts calculated. Results of a representative experiment carried out in triplicate are shown as means and standard deviation. The line indicates a Kd of 23 µM, and has a correlation coefficient of 0.9986. In four independent experiments Kds of 20-50 µM were found. Control experiments with beads alone showed a Kd greater than 10 mM.
Localization of anti-idiotype reactivity
Immunofluorescence with 5D3 on BHK-21 cells maintained at 37 °C (Fig. 7a) yielded a weak punctate staining pattern throughout the cytoplasm, which tended to concentrate in a crescent next to the nucleus. We reasoned that the low level of signal might reflect the fact that the receptor is almost continuously occupied during the normal functioning of the secretory pathway.
FIG 7 Intracellular location of the receptor.
Methanol-fixed NRK cells were labelled with 5D3 culture supernatant
after incubation at 37 °C (a), 2 h at 15°C (b),
2 h at 37 °C in the presence of 10 µg ml-1
brefeldin A (c), after 30 min recovery at 37 °C from
a 2 h brefeldin A block (d). e, Labelling of NRK
cells with mouse polyclonal anti-receptor antiserum. f,
The same cell labelled with a rabbit polyclonal antiserum against
the Golgi apparatus. g Labelling of methanol-fixed untreated
NRK cells with an affinity-purified rabbit polyclonal antiserum
to the soluble receptor fragment. The same field shown in (h)
double labelled with the mouse monoclonal antibody, 1D3, to reveal
the distribution of PDI. The arrow indicates a region of a flattened
cell in which receptor is concentrated in an area from which PDI
is excluded.
METHODS For immunofluorescence NRK cells were grown on multiwell slides, treated as described above, washed briefly in PBS of the appropriate temperature and fixed in methanol at -20 °C for 6 min. The cells were washed twice in PBS and incubated in blocking buffer (PBS containing 10% (v/v) normal goat serum) at room temperature for 30 min in a humidified chamber. First antibody incubations were for 1 h at room temperature. The slides were washed five times with PBS, re-blocked with blocking buffer then exposed to fluorochrome-conjugated second antibody (Zymed Inc.) diluted according to the manufacturer's instructions in blocking buffer for 1 h. After extensive washing in PBS, the slides were mounted with Moviol and examined in a Leitz Axiophot microscope. Photography was with Tri-X rated at 1,600 ASA. Final magnification, X6,300 (all panels).
Treatments that interfere with the secretory pathway might be expected to interfere with recycling to the ER and result in decreased receptor-occupancy and therefore increased reactivity with the antiidiotype antibodies. Incubation at 15°C blocks transport between ER and Golgi and causes elaboration of a novel tubular structure in the peri-Golgi region14. Incubation of BHK-21 cells at 15 °C for 2 hours caused an increase in the intensity of the immunofluorescence obtained after staining with 5D3, together with a tendency for the punctate stain to coalesce into larger strongly labelled regions in the cytoplasm (Fig. 7b). Under the same conditions, anti-KDEL antibodies gave their normal reticular pattern of immunofluorescence (data not shown). Hence, incubation at 15 °C, a treatment that interferes with ER to Golgi transport14, increases the accessibility of the 72K putative receptor to specific anti-idiotype antibodies and alters its localization. This observation associates the receptor with the salvage compartment14.
Brefeldin A has a profound reversible effect on the exocytic pathway, which is manifest as a condensation of cis and medial Golgi into the ER24-25. When NRK cells were treated with brefeldin A for 2 hours the normal Golgi structure was completely dispersed, although the reticular pattern associated with the ER labelling of rabbit anti-KDEL antisera remained essentially unchanged. Under these conditions staining with 5D3 revealed the usual vesicular pattern, but with increased intensity (Fig. 7c). After removal of the brefeldin A, 5D3 showed a reticular ER-like pattern of labelling (Fig. 7d) before returning to the normal staining pattern seen in Fig. 7a. This behaviour also associates the antigen with the salvage compartment25.
Polyclonal antisera, raised in BALB/c mice by immunization with soluble receptor from either 5D3 or 9D6 cells, labelled the 72K band in VG cell lysates (Fig. 2d). These polyclonal antisera showed an immunofluorescent labelling pattern that was indistinguishable from the pattern obtained with 5D3. The widely distributed punctate labelling seen with these reagents showed the same tendency to concentrate to one side of the nucleus (Fig. 7e) and the same appearance in a juxtanuclear tubular compartment after a 15 °C block (data not shown) as observed with the anti idiotype antibody. Staining of the field shown in Fig. 7e with a polyclonal antiserum against the Golgi apparatus gives an overlapping but quite distinct cisternal pattern (Fig. 7f). Double-label immunofluorescence with the polyclonal antiserum to soluble receptor fragment (Fig. 7g) and I D3 (Fig. 7 h ) show that the receptor and its ligand have overlapping but distinct locations.
The anti-idiotypic approach to the identification of a receptor for the KDEL retention signal rests on the assumption that a single receptor is responsible for the recognition of the several KDEL proteins. Consistent with this assumption, three independent approaches led to an anti-idiotypic response against KDEL peptide antibodies and these independently-produced antiidiotypic antibodies all recognized the same 72K transmembrane protein. The third round of immunization provided the strongest support for our initial assumption that one receptor serves several KDEL terminated ligands. Antisera produced in response to immunization with 5D3 cells show a broad specificity for the KDEL signal but do not recognize the nonfunctional K . .. GL tail. A similar effect can be seen in the third-round response generated by production of ascites in non-irradiated mice from either of the anti-idiotypic hybridomas. These responses again show reactivity with several KDEL tail sequences but not with K . . . GL or HDEL peptides. This reactivity justifies the identification of the 72K antigen as a KDEL signal-binding protein and our localization and functional studies are consistent with a role in recognition of the KDEL retention signal in vivo. Henceforth we will refer to the antigen as the receptor.
Both 5D3 and 9D6 hybridomas secrete in large amounts a soluble form of the receptor that is highly glycosylated, oligomeric and complexed with antibody. Labelling experiments show that the production of soluble receptor exceeds antibody production in both hybridomas. Polyclonal antibodies against the secreted soluble receptor from 9D6 and 5D3 cells recognize the normal 72K receptor in VG cells. Therefore the secreted forms share antigenic determinants with the normal receptor, beyond those recognized by 5D3 and 9D6 antibodies. Whether the soluble form results from changes in transcriptional or post-transcriptional events remains to be seen, but we interpret overproduction of soluble receptor as a mechanism to titrate the anti-receptor antibodies in the lumen of the ER. Intact membrane-bound receptor present in the hybridoma cells and protected in this way from the antibody can then function sufficiently to permit survival of the cells.
The epitope recognized by 5D3 and 9D6 seems to be widely conserved throughout evolution. All of the vertebrate cell lines and tissues tested that show reactivity with our anti-tail antibodies (S.F., J.T. and D.V., manuscript in preparation) also react with the anti-idiotype antibodies. None of the other cell lines tested showed the overexpression and secretion of the antigen observed in the 5D3 and 9D6 hybridomas. In normal cells the receptor seems to be a minor membrane protein. Our anti-KDEL tail antibodies show negligible reactivity with a peptide bearing the S. cerevisiae signal (HDEL) or with cell lysates from this yeast. Correspondingly, our anti-idiotype antibodies show no significant reactivity in this organism.
Preliminary studies (results not shown) provide some intriguing insights about the receptor. Western blotting with a battery of our anti-KDEL tail antibodies indicate that the receptor is not itself a KDEL protein. The receptor is an intrinsic membrane protein that spans the membrane at least once (we cannot exclude several membrane-spanning domains). Nonreduced gels of VG lysates indicate the protein may be oligomeric and the antigen precipitated from 9D6 or 5D3 cells is disulphidecrosslinked to form apparent trimers and higher-order oligomers. Finally, the secreted receptor is posttranslationally modified. That the secreted antigens from 5D3 and 9D6 cells, although cross-reactive, migrate at different Mrs reflects their sialylation, as this difference in mobility is abolished by neuraminidase-digestion. The receptor in normal cells does not show this high degree of glycosylation, presumably because it is not exposed to the later stages of the secretory pathway.
The localization of the receptor is central to an understanding of its function in the cell. The patterns of immunofluorescence labelling of NRK cells at 37°C with 5D3, which should be specific for receptor that does not have KDEL ligand bound to it, indicate that the receptor is in a compartment distinct from the ER and the Golgi apparatus but related to them both. The labelling of cells incubated at 15° C establishes that the receptor is in the 15° C compartment, which we equate with the salvage compartment. The patterns of labelling during brefeldin A treatment and recovery from it are also consistent with the proposal that unoccupied receptor is in a compartment between the ER and the Golgi stack, that is, the salvage compartment. Here, the unoccupied receptor is positioned appropriately to capture KDEL proteins that escape from the ER and to recycle them. Results obtained with polyclonal and monoclonal antibodies against isolated receptor show a very similar labelling pattern, suggesting that only low levels of receptor are ever found in other cellular compartments. We intend to investigate the localization of the receptor by EM immunocytochemistry.
The binding of a recycling receptor to KDEL tails must be modulatable. The receptor must have a high affinity for KDEL tails when it captures them and a lower affinity under the conditions of the ER where they are released. The secretion of a soluble form by the hybridomas allowed us to isolate receptor for binding studies. Binding of this material to peptide columns demonstrated that it specifically recognized the KDEL terminated peptides rather than a K... GL peptide. This specific binding was strongest near physiological pH and was divalent cation-dependent. Pure labelled PDI bound to isolated receptor with a Kd of ~23 µM. Although post translational modifications of the secreted receptor may cause a change in the characteristics of binding, this Kd is within the physiological range. The concentration of KDEL tails calculated for the ER lumen is well above this constant, indicating that our binding protein is a plausible candidate for the physiological receptor. In addition, this value suggests that the difference between the high-affinity (salvage compartment) and low-affinity (ER) forms need not be large. The transferrin receptor, for example, has only a 20-fold greater affinity for diferritransferrin than for apotransferrin26. Such a change in the affinity of our putative receptor would allow it to function in the recycling and release of KDEL proteins into the ER lumen, where their concentration is high. The size of this affinity constant also provides an explanation for the difficulty experienced by us, and others, in isolating a KDEL receptor by conventional affinity methods. Finally, the conclusion that receptor binding is divalent cation dependent is supported by recent work27 that suggests the involvement of Ca2+ in the retention of KDEL-terminated proteins in the ER in vivo. Furthermore, we have observed that the availability of cellular receptor for binding to anti-idiotypic antibody in immunofluorescence experiments decreased after incubation of cells in medium containing the calcium ionophore A23187 and Ca2+ (S.F., J.T. and D.V., manuscript in preparation).
This work has identified a KDEL binding protein that we think
is a good candidate for the physiological receptor. In summary,
our evidence that the 72K protein is indeed the receptor comprises
the following: (1) separate routes beginning with different KDEL
sequences identify the same oligomeric transmembrane antigen;
(2) a broadened specificity is observed in the third-round antibodies;
(3) the antigen is concentrated in a compartment intermediate
between ER and Golgi; (4) the KDEL binding site lies in the lumen
of the secretory pathway; (5) the anti-idiotype hybridomas dramatically
upregulate this protein; (6) isolated antigen binds specifically
to peptides containing the retention signal and (7) isolated antigen
binds physiological ligand with a Kd compatible with
a recycling model for ER retention. Although it remains formally
possible that the localization and binding properties of this
protein are only coincidentally those expected of a KDEL receptor,
the properties of this protein are consistent with the model of
Munro and Pelham10, and indicate additional features
of ER retention. The binding site of one receptor species can
accommodate all known KDEL signals, hence several receptors are
not necessary. The receptor is a minor transmembrane protein,
which must therefore operate by a recycling mechanism. The receptor
is not normally detectable within the bulk of the ER or the Golgi
apparatus. The receptor appears restricted to a relatively small
region that may represent the exit sites from the ER and the return
sites for captured ligands. Both 15°C incubation and brefeldin
A treatment cause changes in receptor localization consistent
with it being predominantly present in the intermediate or salvage
compartment. The observed Kd of receptor for ligand
would allow the receptor to capture ligand in the salvage compartment
and release it into the ER without requiring large changes in
affinity constant. The moderate changes necessary could be mediated
by an environmental factor such as pH or divalent cation concentration.
Whether the receptor recycles continuously or only when occupied
by ligand is unclear; but we note that not only is the receptor
a transmembrane protein but it and several of its ligands are
oligomeric3. This opens the possibility that the receptor
signals its occupancy to the cytoplasm by clustering28.
Received 22 December 1989: accepted 27 April 1990.
1. Blobel, G. & Dobberstein, B. J Cell Biol. 67,
2. Palade, G. Science 189, 347-358 (1975).
3. Pelham, H R. B. A. Rev. Cell Biol. 5,1-24 (1989)
4. Warren, G. Nature 327, 17-18 (1987).
5. Haas, I. G. & Wabl, M Nature 306, 387-389 (1983)
6. Freedman, R B. & Hillson, D. A Enzymology of Post-translational Modification of Proteins157-212 (Academic, London, 1980).
7. Freedman, R. B Trends Biochem. Sci. 9, 438-441(1984),
8. Weibel, E. R., Staubh, W., Gnagi, H. R & Hess, F A J. Cell Biol. 42, 68-91(1969).
9. Munro. S. & Pelham, H R B. Cell 46, 291-300 (1986)
10. Munro, S & Pelham, H. R. B. Cell 4S, 899-907 (1987)
11. Pelham, H R. B., Hardwich, K. G & Lewis, M J. Embo J. 7,1757-1762 (1988)
12. Ceriotti, A & Colman, A. EMBO J. 7, 633-638 (1983).
13 Pelham H. R B. EMBO J. 7, 913-9l8 (1988)
14 Saraste, J. & Kuismanen. E. Cell 38, 535-549 (1984)
15. Tooze. J., Tooze, S. & Warren. G Eur J. Cell Biol. 33, 2B1 293 (1984)
16. Balch W E, Elliott, M. E. & KeMer, D S. J. Biol. Chem. 261,14681-14689 (1986).
17. Gaulton, G. N & Greene, M. L. A. Rev. Immun. 4, 253-280 (1986).
18. Vaux, D J. T, Helenius, A & Mellman, I. Nature 332, 36-42 (1988).
19 Farid. N R. & Lo, T C. Y. Endocr. Rev. 6,1-23 (1985).
20 Pain, D., Kanwar, Y S. & Blöbel, G. Nature 331, 232-237 (1988).
21 Tooze, J, Kern. H F, Fuller. S D & Howell. K E J. Cell Biol. 109, 35-50 (1989).
22. Smith, M. J. & Koch, G. L. E. EMBO J. S, 3581-3586 (1989).
23 Bordier, C. J. Biol. Chem. 256,1604-1607 (1981).
24. Misumi, Y. et al. J. Biol. Chem. 261,11398-11403 (1986).
25. Lippincott-Schwertz, J. et al. Cell 60, 821-836 (1990).
26. Klausner, R. D. et al. Proc. Natn. Acad. Sci. USA. S0, 2263-2266 (1983). 27 Booth, C. & Koch, G. L. E. Cell 59, 729-737 (1989).
28 Fulier, S. D Cell 45, 923-934 (1987).
29 Frank, R. & Gausepohl, H. in Modern Methods in Protein Chemistry Vol. 3 (ed. Tschesche, H ) 42-60 (De Gruyter, Berlin & New York).
30. Laemmli, U. K. Nature 227, 680-685 (1970).
31. Johnson, D. A. et al. Gene Analyt. Techn. 1, 3-8 (1984).
32. Lambert, N. & Freedman, R. B. Biochem. 1213, 225-234 (1983).
33. Fisher. P et al. J. Cell Biol. 92, 674-686 (1982).
34 Fazekas de St Groth, S. & Scheidegger, D. J. Immun. Methods 35,1-21 (198O).
35 Kreis T. EMBO J. 15, 931-941 (1986).
36. Hemmila, I. et al. Analyt. Biochem. 137, 335-343 (1984).
37. Vaux, D J T & Gordon, S. J. Cell Sci. 77,109-127 (1985).
ACKNOWLEDGEMENTS. We acknowledge the technical assistance and forebearance of P Buck and M. Kail. We thank our colleagues R. Frank and H. Gausepohl for the synthesis of the peptides and for useful advice on peptide chemistry. We thank B. Burke for the gift of a polyclonal antiserum against a protein of the Golgi apparatus. We also thank P. Walter and H. Pelham for their comments and encouragement.
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© Copyright 2000 Department of Biology, Davidson College, Davidson, NC 28036
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2017 (Senior)
Appeared in 41 games, making 34 starts at designated player and first base ... posted a .206 batting average with three doubles, four home runs, 13 RBis and 10 runs scored ... drew 14 walks and a pair of HBPs ... went 2-for-2 with an RBI against Rider (Feb. 17) ... had a 3-for-4 day with a home run, two RBIs and a run scored against Delaware (Mar. 10) ... went 2-for-3 with a double and three RBIs as well as a pair of walks against Campbell (Mar. 11) ... went 2-for-4 with a solo home run against Appalachian State (Mar. 22).
2016 (Junior)
Was the only Spartan to play and start in all 59 games … batted .273 (47-for-172) with 35 RBI, seven home runs and 11 doubles … named to the Preseason All-SoCon First Team … recorded 426 putouts at first base with four assists and five errors for a .989 fielding percentage … led the team with 19 walks … recorded her first multi-hit game of the season against No. 25 NC State (Feb. 13) … had 12 multi-hit games and 10 multi-RBI games … compiled a nine-game hit streak, the longest on the team, and reached base safely in nine consecutive games as well … tallied three three-hit games and one four-RBI game … tied a career-high two doubles against Iona (Feb. 27) … stole her first career base against Bryant (Mar. 15) … recorded a career-high 14 putouts against Iona (Feb. 27) … tied her career-high for runs scored against Coastal Carolina (Feb. 12) … finished the season with 18 extra base hits.
Saw action in 43 games, starting 43 at first base ... selected SoCon first team All-Conference at first base ... set career highs in average (.366), runs (22), hits (45), doubles (9), home runs (8), RBI (28), slugging percentage (.634), walks (27) ... led the team with 322 putouts at first base ... had 11 multiple hit games with two three hit games against Bryant (March 11) and against Western Carolina (May 7) in the final game of the season at the SoCon Tournament ... had a seven-game hitting streak from April 11 through April 28 ... drove in multiple runs in seven games, including a season-best four off a grand slam at Mercer (April 11) ... hit a home run against ACC member NC State (March 18) ... had two home runs in a three-game series win at Furman (May 2-3) ... collected four hits in the SoCon Tournament, including a home run against Western Carolina (May 7).
2014 (Freshman)
Played in 48 games, starting 47 in the outfield and first base ... hit .267 on 36 hits, 17 runs scored, eight doubles, three home runs and 22 RBI ... was 1-for-3 with an RBI double against Presbyterian (Feb. 9) ... went 2-for-4 with a home run and two RBI against Army (Feb. 21) ... picked up two hits, two RBI and a double against Hampton (Feb. 23) ... had a 3-for-4 effort with two doubles and two RBI against UNCW (Feb. 28) ... had a 10 game hitting streak from Feb. 23 through Mar. 15 ... went 2-for-4 with a run scored against North Carolina A&T (Mar. 5) ... was 3-for-4 with a double, two RBI and three runs scored in game two against Gardner-Webb (Mar. 11) ... picked up runs batted in against Samford (Mar. 22) and North Carolina A&T (Mar. 27) ... went 1-for-2 in the series finale at Furman (April 6) ... had a RBI in game two against Charlotte (April 23) ... hit a two-run home run in the series finale at Appalachian State (April 27) ... finished the season with a stellar SoCon tournament, hitting a key three-run home run against Georgia Southern (May 9) in the semifinals ... named to the SoCon All-Tournament team after picking up six hits in the tourney.
High school
a first baseman for Dominion High School in Sterling, Va. that earned first-team all-district and all-region honors in 2012 … also is a two-time Coaches Award winner in 2011 and 2012 … finished the 2012 season with a .516 batting average, .600 on base percentage and an impressive .981 fielding percentage … has a 3.83 GPA and is a member of the National Honor Society and Spanish Honor Society … She plays for on the Ashburn Shooting Stars 18U Gold club team in Ashburn, Va.
Victoria "Tory" Wright ... born July 24, 1995 in Washington, D.C. … daughter of Robert and Nancy Wright … has a brother Evan Wright who attends South Carolina University … major is undecided.
|First Base/Outfield
|Potomac Falls, Va.
|High School:


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Brief SummaryRead full entry
StreptococcusStreptococcus is a genus of spherical Gram-positive bacteria belonging to the phylum Firmicutes and the lactic acid bacteria group. Cellular division occurs along a single axis in these bacteria, and thus they grow in chains or pairs, hence the name — from Greek στρεπτος streptos, meaning easily bent or twisted, like a chain (twisted chain). Contrast this with staphylococci, which divide along multiple axes and generate grape-like clusters of cells. Most streptococci are oxidase- and catalase-negative, and many are facultative anaerobes.


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Through GALILEO, Georgia citizens have access to authoritative, subscription-only information that isn't available through free search engines or Web directories. Here's a sampling of what's inside:
Select your institution below to get the password from your GIL account or to log in with your institutional credentials.
(See also "Private College or University" below if your school is not in the list above.)
Note: To obtain your GALILEO password, you will need to log into your GIL account. Follow these instructions to obtain your GALILEO password:
For more information on obtaining your GALILEO password, see this FAQ.
GALILEO Guest access includes non-password-protected resources only, such as the Digital Library of Georgia databases, the New Georgia Encyclopedia, and other public databases. It is possible to log in as a Guest in one of three ways:


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St. Pete Beach is a barrier island in Pinellas county with award-winning, beautiful beaches on the Gulf of Mexico. With miles of pristine white sand, St. Pete Beach offers visitors a variety of views, activities and Florida sun.
St. Pete Beach is nestled on Long Key. Upham Beach, located on the northern tip of St. Pete Beach, is one of the few spots along the Gulf of Mexico known for surfing. Playground and showers are available. Beach access is available through Pinellas County Parks or at one of several beachfront resorts. The St. Pete Beach facility offers plenty of parking, restrooms, showers, a water fountain, and three boardwalks to carry visitors over natural sand dunes and sea oats to a white sandy swim area and a spectacular view of the Gulf of Mexico.
Accommodations range from world-renowned resort to mom-and-pop motels, to quaint bed and breakfasts. There is the architecturally and historically grand Don CeSar Resort, which offers its guests a luxurious view of the gulf amidst the grounds of the historic "Pink Lady." For a more expansive experience, the TradeWinds Islands Resort offers its guests endless beach activities, 14 restaurants, entertainment venues, or just relaxing by one of their seven pools.
For fun on the water, there are several marinas in St. Pete Beach offering boat rentals, parasailing, deep sea fishing, dolphin watches, and more. Beyond the surf visitors can spend the day shopping on historic Corey Avenue, dine at a waterfront restaurant, or spend the day skim boarding.
Other attractions in the area include several world-class museums, including the Salvador Dali Museum--which offers the most comprehensive collection in the world devoted to the Spanish master--and the Florida International Museum--which hosts national and international traveling exhibitions. The Pier, a five-story dining and shopping complex.
For more information on St. Pete Beach visit http://www.visitstpeteclearwater.com/articles/st-pete-beach.


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Florida's defense was the strongest, most dependable, most confident aspect of the team, but it disappeared two weeks ago in Missouri.
Many strange and unexplainable things have befallen the Florida Gators this season, from Jeff Driskel's broken leg, to the numerous non-contact ACL injuries, to the failing offense.
But maybe the hardest thing of all to explain is the disappearance of the defense two weeks ago in Missouri. The strongest, most dependable, most confident aspect of this team looked dazed and confused in an epic meltdown.
It was unexpected. It was shocking.
"Yeah it was," junior safety Cody Riggs said. "We made a lot of mistakes. A lot of things we let happen, we shouldn't have let happen. We've got to correct that. We don't have any excuses. We just played really bad. We can't blame it on the offense. We didn't put them in good positions. They had bad field position pretty much the entire game. It's up to us to give them good field position so they can score points.
"We just didn't play well. Coaches put us in position to make plays. We didn't make the plays we were supposed to make."
For the defense, its bad day started on the first play of the game, and it involved Riggs.
On a 41-yard pass completion to L'Damian Washington, Riggs' high hit on the wide receiver was ruled targeting and he was ejected from the game. On the next play, the Tigers beat the secondary for a 20-yard touchdown pass.
The tone had been set. The rout of the UF defense was on.
The Tigers went on to hit big play after big play (on the ground and through the air). When it was mercifully over, the carnage was immense: 36 points and 500 yards surrendered by a Florida defense that came into the game leading the SEC in seven statistical categories.
"It was shocking, but we can only move forward and focus on the next game, which is Georgia now and just try to keep that total yards below that," junior cornerback Marcus Roberson said.
The Gators, now really feeling the loss of star defensive tackle Dominique Easley, gave up 205 rushing yards and surrendered an uncharacteristic big play on the ground, a 50-yard run that set up a momentum-changing touchdown in the second half.
The secondary was also burned by big plays, giving up receptions of 52, 41, 27 and 20 yards from a redshirt freshman quarterback (Maty Mauk) making his first start.
"Shocked by the performance? I don't know, I think there were obviously things we could have done much better in that game," defensive coordinator D.J. Durkin said Wednesday. "They hit us on some plays. We needed to bounce back quicker.
"We're going to get hit on a play here or there, especially if you're playing receivers like they've got. We've just got to line back up and go play. And I think at some point we didn't react the way we needed to on defense. That's something that hasn't happened with our group, and obviously we hope it doesn't happen again."
Durkin said he did not see the defensive players hanging their heads during the rout — or during a bye week last week when the tape was being broken down and corrections made.
"I think our guys are resilient," he said. "They work hard, and they're confident in what we're doing. I think you'll see it (against Georgia).
"What makes me confident it won't happen again? The way our guys practice, the way they prepare. I think we've got guys with great character and guys that are really good football players.
"So, their response these past two weeks and how they're practicing and preparing tells me that you move on. They know what it takes and what you need to do."
Junior linebacker Michael Taylor said the Gators will not give up another 500-yard game. In fact, he guarantees it.
"Very safe to say (that won't happen again)," Taylor said. "That's not characteristic of us. That's not what we allow and that's a one-time thing. I can guarantee you that won't happen again."
Defensive end/outside linebacker Dante Fowler Jr. said the poor performance against Missouri has shaken the Gators' confidence, but not broken it.
"I feel like our defense is still a great defense," he said. "Once you lose a game or two, you kind of lose your confidence. And all we've got to do is just get our swagger back. I feel like once we get our swagger back, and get our confidence back up, we're going to be back to how we were at the beginning of the season."
Contact Robbie Andreu at 352-374-5022 or email@example.com. Also check out Andreu's blog at Gatorsports.com.


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A 35-year-old man has been charged with assaulting a 19-year-old man with a machete, according to an arrest warrant.
A 35-year-old man has been charged with assaulting a 19-year-old man with a machete, according to an arrest warrant.
Pedro Lopez Tabares of 511 Kildee Drive has been charged with felony assault with a deadly weapon inflicting serious injury and driving while license revoked. He was placed in the Davidson County Detention Center under a $1,000 secured bond and has a court date of Aug. 19.
The Lexington Police Department received a report of the incident at 5:05 p.m. Saturday.
Michael McIntosh reported an assault at Tabares' residence, according to an arrest report. He reported that Tabares cut his hand with a machete in front of the residence. McIntosh was taken to Wake Forest Baptist Health — Lexington Medical Center for treatment of the cut on his left hand.
Capt. Robby Rummage of the LPD said the two men were known to each other, and the incident stemmed over an argument involving property damage of a vehicle. The captain confirmed McIntosh's injury was not life-threatening.
McIntosh was treated and released, according to a hospital spokesman.


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Kristina Rogers
Kristina is an experienced senior leader in the areas of strategic marketing, corporate strategy, and global business development. Her areas of depth and expertise include new concept and offer development, market entry strategy, strategic category management, profitable growth planning, strategic marketing, and market evolution planning in both the B2C and B2B marketplaces.
As Global Sector Leader for Consumer Products and Retail, Kristina is responsible for shaping the strategy and enabling the implementation of EY's go-to-market strategy in the sector. Prior to her current role, she served as Emerging Markets Leader for Consumer Products and Retail.
Before joining EY, the Harvard Business School alumnus was a Senior Partner at Monitor Group, a global strategy consulting firm, co-leading their Middle East Management Team.
Kristina is an ambassador for EY's global approach having lived and worked in more than 20 countries, including Canada, the US, the UK, the UAE, South Africa and Turkey, where she currently resides. Over the course of her career, Kristina has had long-term client relationships with some of the world's largest global consumer products companies including Coca Cola, Cadbury, Procter & Gamble, and Shell (retail forecourts) helping them to solve some of their most complex strategic marketing issues across geographies.
Kristina is currently a member of the Financial Times' 'Global Board Ready Women' group and was chosen as one of 40 high-potential women by Forbes Magazine in Turkey to join the 'More Women on Boards' program. Kristina currently sits on the Corporate Marketing Advisory Board at Medtronic.
Kristina received an Honours Bachelor of Commerce degree with High Distinction from Queen's University in Canada and an MBA degree from Harvard University.
In her spare time, Kristina enjoys photography, scuba diving. and Canadian fiction. She is a mother of two – a daughter 11, and a son 8.
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Letter to the Editor, Aug. 31, 2013: Open Letter to Mayor and City Council.
Letter to the Ridgecreset Mayor and the Council
Attn Mayor and Council,
I read in the paper that the Mayor has tasked the Planning Commission to look into the idea of Ridgecrest becoming a charter city. I question the authority the mayor used to task the Manager and Planning Commissioners to do this.
Individually the mayor and council has NO authority to task the staff to do anything. They can only task at a duly formed council meeting and then by a concurrence by at least a simple majority to task staff to do anything.
Other than the mayor talking about this when he first got elected, I do not remember ANY council meeting where this was brought as an agenda item for discussion. And I do not remember any council vote on the issue either. I would like them to state for the public the date this topic was agendized and then discussed with public input and then an action taken by the council to task the Planning Commission to do this.
Further, the idea of becoming a charter city is not in the best interest of Ridgecrest. I am not in favor of this at all. And I don't believe we should be assigning this very time consuming task to city staff or the Planning Commission unless there was at least a public airing of the concept at a council meeting.
Reasons against it:
1. Will make Ridgecrest a taxing authority.
2. Will abolish term limits for the mayor and council.
3. Will make the mayor position very powerful and high paid.
4. Will be very costly to establish. Typical cost to become a charter city is at least $100,000 in just legal and consultant fees.
Come on council. Let's have a public discussion before wasting staff and Planning Commission time.
Thomas Wiknich


text:
From Imperial Wiki
Versus usually, but not always, refers to the debate between SW and ST. Covering such topics as: Which franchise has faster ships? Which franchise has better technology? Which side would win in an total war scenario?
The Versus debate has been dead for sometime. The ICS settled the debate quite thoroughly, although there are some who insist that the ICS is not canon or is flawed.
Other Versus
The Versus, however, is not limited to only SW vs ST, but can be applied to any matching between any science fiction franchises, from fairly balanced matches, such as Firefly versus neo Battlestar Galactica to outrageously unfair matches, such as Star Trek versus the Xeelee.


text:
The Motorcycle Industry Council's Business Advisory & Forecast group has issued a report that predicts a sales decline in the US new motorcycle market for 2012. The news comes fresh on the heels of the 0.3% gain that the motorcycle industry's leading brands experienced in 2011 in the American market, and is the first time that the MIC has forecasted future new motorcycle sales for the United States.
Adding some validity to the report is the fact that the MIC, in conjunction with the Institute for Trend Research, accurately predicted 2011's modest sales growth. This news is interesting to note, as it goes counter to news about the recovering economy and the increased national average gasoline price, both of which have been linked to previous bumps in volume for motorcycle sales.


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The first shipments of cellulosic ethanol produced at a commercial scale should be available soon according INEOS Bio, which owns the facility in Florida that is making the next generation fuel. The company said late last month that its Indian River BioEnergy Center is now generating cellulosic ethanol at commercial pace using gasification and fermentation technology applied to vegetative and wood waste. It will ultimately have an annual output of eight million gallons of cellulosic ethanol and six megawatts of renewable power.
Wheat straw and biomass grown on marginal land or even conservation program acres could be excellent feedstocks for cellulosic ethanol, producing a new source of income for farmers and clean, renewable fuel to offset America's total oil consumption. More about the INEOS facility's milestone is at http://www.ineos.com/en/businesses/INEOS-Bio/News/INEOS-Bio-Produces-Cellulosic-Ethanol/.