It is generally recognized that there is a need for improved teaching of nutrition in medical schools and for increased education of the general population. A questionnaire, derived in part from a study of physician knowledge, was administered to first year medical students in order to assess their knowledge of various aspects of nutrition and metabolism, and as a teaching tool to transmit information about the subject. The performance of first year students was consistent with a generally educated population but there were surprising deficits in some fundamental areas of nutrition. Results of the questionnaire are informative about student knowledge, and immediate reinforcement from a questionnaire may provide a useful teaching tool. In addition, some of the subject matter can serve as a springboard for discussion of critical issues in nutrition such as obesity and markers for cardiovascular disease. A major barrier to improved teaching of nutrition is the lack of agreement on some of these critical issues and there are apparent inconsistencies in recommendations of government and health agencies. It seems reasonable that improved teaching should address the lack of knowledge of nutrition, rather than knowledge of official guidelines. Student awareness of factual information should be the primary goal. Like many medical schools, SUNY Downstate Medical Center has been trying to improve the teaching of nutrition in the curriculum. The study presented here had two goals. First, we wanted to assess the knowledge of the first year class on the subject and, second, we hoped to use the questionnaire method, with immediate feedback, as a mechanism for imparting information on the areas covered. Whereas some form of nutrition education is part of the curriculum of most medical schools , it is gPerhaps the most important problem in introducing nutrition into the medical curriculum is the lack of agreement on what should be taught. There is strong, even contentious debate about the most fundamental issues such as obesity, cardiovascular disease, and diabetes. Thus, concerns about inadequate physician knowledge frequently refer to ability to counsel patients according to standard guidelines -6. Here The quiz is presented twice: first, as given to medical students, and then, a second time, with the answers and the comments given to students immediately upon completion of the quiz. A follow-up email that was sent to students after results were tabulated has also been reproduced. Results and Discussion provides data on student performance and additional discussion.et al. [The following is a verbatim reproduction (with addition of references) of the questionnaire given to first year medical students at SUNY Downstate Medical Center. The first four questions were taken from Flynn, et al. . FollowiThis questionnaire is anonymous and does not affect your grade. Make up a User Name (in case there is a follow-up) that only you know and that is not common, like Sherlock-37)User Name _________________________________________________The questionnaire is designed to test your general knowledge, not anything you have learned in the course.1. A good source of monounsaturated fat is: _____ Butter_____ Canola Oil_____ Corn Oil_____ Flaxseed Oil_____ Olive Oil_____ Safflower Oil_____ Soybean Oil_____ don't know2. The diet component that is most likely to raise triglycerides is (select one)_____ Fat_____ Carbohydrate_____ Protein_____ don't know3. In general, what effect does a low-fat diet have on triglycerides?_____ Increase_____ Decrease_____ no change_____ don't know4. In general, what effect does a low-fat diet have on HDL-c (high density lipoprotein-cholesterol) ?_____ Increase_____ Decrease_____ No change_____ Don't know5. In the past thirty years, the per cent fat in the American Diet has:_____ Increased_____ Decreased_____ Stayed about the same.6. The most energy dense food  is:_____ Carbohydrate._____ Protein._____ Fat.7. High total blood cholesterol can be lowered significantly by:_____ Diet_____ Drugs such as statins_____ Diet or drugs are equally effective_____ Neitherincrease the risk of cardiovascular disease:8. The dietary change that is most likely to _____ unsaturated fat → saturated fat _____ unsaturated fat → carbohydrate_____ carbohydrate → unsaturated fat_____ carbohydrate → saturated fat_____ saturated fat → carbohydrate_____ saturated fat → unsaturated fat9. Glycemic Index measures the increase in blood sugar over 2 hours per gram of carbohydrate ingested, compared to glucose (=100). For each food indicate the approximate glycemic index as: H, high (70–100), M, Medium (40–70) or L, Low (< 40). You may enter a number if you think you know or can figure it out:_____ white bread_____ whole wheat bread_____ ice cream_____ carrots_____ sucrose (table sugar)_____ fructose_____ bran muffin_____ banana10. The substances in the following list that either are themselves or are considered to contain large amounts of complex carbohydrates._____ white bread_____ whole wheat bread_____ ice cream_____ fructose_____ sucrose (table sugar)_____ corn starch_____ fiber11. In the first list check the vitamins that are generally considered to have antioxidant activity. In the second list check the vitamins that are precursors for oxidative coenzymes. ANTIOXIDANTS_____ ascorbic acid (vitamin C)_____ niacin_____ riboflavin_____ thiamine6)_____ pyridoxal phosphate  that reported the results of a questionnaire designed to assess the level of knowledge of physicians. The authors were critical of the responses; the paper was entitled "Inadequate physician knowledge of the effects of diet on blood lipids and lipoproteins." In particular, "Physicians showed a poor understanding of the effects of changing the relative intake of carbohydrates and fats on triglycerides and HDL."The first four questions appeared in a recent paper . The ATP III, compared to previous version, advocates lowering triglycerides as a secondary target to lowering LDL. The major conclusions:The rationale for the study were guidelines recommended by the Third Report of the National Cholesterol Education Program Adult Treatment Program  did not know that a low-fat diet, in general, would increase blood triglycerides.• Approximately three-quarters (70% of cardiologists vs. 77% of internists) did not know a low-fat diet would decrease HDL; almost half (45%) thought that a low-fat diet would not change HDL.• About one-half  did not know carbohydrate was the diet component most likely to raise triglycerides.1. A good source of monounsaturated fat is: _____ ButterX _ Canola Oil_ _____ Corn Oil_____ Flaxseed OilX _ Olive Oil_ _____ Safflower Oil_____ Soybean Oil_____ don't knowIt is generally considered that monounsaturated fats are protective of cardiovascular disease (as in the Mediterranean Diet). The effect of monounsaturated fat explains the anomaly in the so called Seven Countries study  which laSome people may be surprised when they actually look at the data Figure . High cois a plant in the Brassica family) and is the subject of popular controversy because of the possible production of trans-fatty acids during processing.You should also know that canola oil is named for CANadian OiL (there is no canola tree) and is a highly processed version of rapeseed oil (which 2. The diet component that is most likely to raise triglycerides is (select one)_____ FatX _ Carbohydrate_ _____ Protein_____ don't know3. In general, what effect does a low-fat diet have on triglycerides?X _ Increase_ _____ Decrease_____ no change_____ don't knowThe phenomenon of carbohydrate-induced hypertriglyceridemia is well established in the literature -15. It i4. In general, what effect does a low-fat diet have on HDL-c (high density lipoprotein-cholesterol)?_____ IncreaseX _ Decrease_ _____ No change_____ Don't know5. In the past thirty years the per cent fat in the American Diet has:_____ IncreasedX _ Decreased_ _____ Stayed about the same.trans fats) is being consumed although no effect of fat per se on obesity, independent of calories, has been found.This is the major point of attack on current guidelines since the obesity epidemic correlates with a decreased percentage of fat in the diet  and increased carbohydrate consumption ,24. Defe6. The most energy dense food  is:_____ Carbohydrate._____ Protein.X _ Fat._ The operational numbers in kcals/g are 4, 4, and 9 for carbohydrate, protein, and fat. This is the basis of traditional recommendations for low fat diets for obesity. The reduction in percentage of dietary fat and the obesity epidemic noted above, however, suggests that this is not a good universal principle. The role of macronutrient composition on satiety, taste, total consumption and effect on weight loss is largely unknown due to the multiple factors and individual differences, and some experimental support can be found for just about any idea.7. High total blood cholesterol can be lowered most significantly by:_____ DietX _ Drugs such as statins_ _____ Don't know_____ Diet or drugs are equally effective_____ NeitherDrugs such as statins (HMGCoA reductase inhibitors) are very effective at reducing cholesterol. Diet can also be effective but usually less so. Combination diet and drugs, however, may be most effective but there is, again, not universal agreement as to what that diet should be.increase the risk of cardiovascular disease:8. The dietary change that is most likely to _____ unsaturated fat → saturated fat X _ unsaturated fat → carbohydrate_ _____ carbohydrate → unsaturated fat_____ carbohydrate → saturated fat_____ saturated fat → carbohydrate_____ saturated fat → unsaturated fatIn addition to the effect on risk factors, epidemiologic evidence suggests that replacing fat with carbohydrate is deleterious. Replacing unsaturated fat with saturated fat will also increase the risk of cardiovascular disease ,9,14,25.9. Glycemic Index (GI) measures increase in blood sugar over 2 hrs per gram of carbohydrate, compared to glucose (=100). For each food indicate the approximate glycemic index as: H, high (60–100), M, Medium (40–60) or L, Low (< 40). You may enter a number if you think you know it:H (70) white breadM (52) whole wheat breadM (50) ice creamM (47) carrotsH (70) sucrose (table sugar)L (20) fructoseHM (60) bran muffinM (50) bananaThe GI is a very rough indicator of rise in blood sugar and is influenced by absorption and the concentration of glucose. Fructose has a low GI (20) indicating slow conversion to glucose in 2 hrs but far from being considered a "good" sugar, at high levels may be very deleterious. The concept of glycemic load (GL) which corrects for the amount of carbohydrate per serving may be a better parameter but runs into problems about serving size. So muffins and candy bars have GL = 15 and carrots only 3 but 80 g of carrots may not be a lot for some people -28.10. The substances in the following list that either are themselves or are considered to contain large amounts of complex carbohydrates.? _ white bread_ ? _ whole wheat bread_ NO ice cream_ NO fructose_ NO sucrose (Table Sugar)_ ? _ corn starch_ ? _ fiber_ If you had trouble with this, it is because nobody knows how the term should be used and, in fact, we recommend it not be used at all. The original chemical definition – to some extent still used in organic chemistry – is that a complex carbohydrate is a polysaccharide  or di- ). Any starch, e.g. corn starch or white bread (poly-glucose: amylose or amylopectin) fits the definition, and it used to be nutritional dogma that, at least in terms of raising blood glucose, complex carbohydrates  were better than simple sugars. When the dogma was finally tested this turned out not to be true and the concept of glycemic index arose. It is clear from question 9 that white bread is nutritionally similar to pure glucose. Probably because of the evocative nature of the word, the term "complex" is still used. Sometimes it means foods that have a low glycemic index due to poor absorption usually due to the presence of fiber, but it is never precise. When people say complex carbohydrate they usually mean the carbohydrate recommended in their diet and missing from somebody else's diet. Suggested nomenclature is "polysaccharides, starch, high fiber," although fiber itself is a heterogeneous category.11. In the first list check the vitamins that are generally considered to have antioxidant activity. In the second list check the vitamins that are precursors for oxidative coenzymes. ANTIOXIDANTSX _ ascorbic acid (vitamin C)_ _____ niacin_____ riboflavin_____ thiamine6)_____ pyridoxal phosphate (Vitamin B12_____ vitamin B_____ vitamin DX _ vitamin E_ REDOX PRECURSORS_____ ascorbic acidX _ niacin_ X _ riboflavin_ X _ thiamine_ 6)_____ pyridoxal phosphate . Megaloblastic anemia can also be caused by a B12. First, vitamin B12 is a cofactor in formation of the amino acid methionine from homocysteine and the folic acid derivative, methyl-tetrahydrofolic acid (methyl-THF): There are only two reactions in humans requiring vitamin BHomocysteine + methyl-THF → Methionine + THF This explains the relation between dietary folic acid and high homocysteine which is a marker for cardiovascular disease and potential birth defects.12, will prevent this reaction from occurring. The effect of B12 deficiency on folic acid is indirect: if methionine synthesis cannot be carried out, methyl-THF will build up ("methyl trap"). This is effectively a folic acid deficiency and anemia is the outcome. The second requirement for B12 involves organic acids and deficiencies can lead to neurologic damage. The anemia in a B12 deficiency may be successfully treated with folic acid, swamping out the methyl trap. Neurologic damage, however, may still occur unless the B12 deficiency is also treated. A dietary deficiency of B12 is rarely seen since little is needed and it is stored well. Deficiency is usually detected in the elderly due to decreased production of intrinsic factor, a protein required for absorption.A deficiency in folic acid or the cofactor, B12 deficiency could be masked. Since the major deficiency is not dietary but absorptive, the problem can't be solved by simply adding B12 as well.The considerations above bear on the recommendation to add folic acid to manufactured food. Critics point out that by preventing anemia, a BThe following analysis was sent as an Email to students after the scores on the quiz were tabulated.Nutrition Journal 2: 19) that reported the results of a questionnaire designed to assess the level of knowledge of physicians. Their conclusion was that "Physicians showed a poor understanding of the effects of changing the relative intake of carbohydrates and fats on triglycerides and HDL."As indicated in the answer sheet, the first four questions are taken from a recent paper  Interestingly the rationale for the study was determining "Physicians' ability to effectively counsel patients with elevated cholesterol to initiate a Therapeutic Lifestyle Changes Diet (TLC)"  which recommends lowering triglycerides as a secondary target to lowering LDL. The TLC Diet recommends total fat as 25–35 % and carbohydrate at 50–60 %. Paradoxically, Flynn, et al.'s assessment of physician knowledge focused on the deleterious effect of carbohydrate on triglycerides. Given this association, it is puzzling that ATP III would council people trying to lower triglycerides to undertake such a diet.In any case, your performance compared to their sample is shown in Figure Two questions of importance:Question 5:In the past thirty years, the per cent fat in the American diet has declined by about 10 %, close to the target level of 30% of total calories set back in the 70's. In order to explain why this has been associated with an obesity epidemic, many have blamed portion-size, the fast-food industry and consumers themselves. In any case, most people did not know that percent fat consumption had gone down.Answers were:Increased: 76 %Decreased 22 %Same 3 %Question 6. Again, it was surprising that so many people did not know the relative energy density. The answers:Carbohydrate 19 %Protein 7 %Fat 74 %). The discrimination coefficient varies between -1 and +1 and measures the extent to which performance on a particular question reflects performance on the quiz as a whole, that is, whether a question discriminates high performers from low performers. A typically high value of +0.4 means that the question was answered correctly by most students who did well on the exam and answered incorrectly by most who did not.Questionnaires were collected from 111 students, all but 6 of whom answered all the questions. Student answers were tabulated and discrimination coefficients were determined by point biserial correlations  using LXBecause the questions had different goals (knowledge assessment vs. motivation for accepting new information), and because some (e.g. questions 2–4) are interrelated, no measure of performance or statistics were carried out for the quiz as a whole.et al. used four questions that succinctly identified both practical and conceptual knowledge bearing on the ability to implement dietary recommendations from the Adult Training Program (ATP III) of the National Cholesterol Education Program (NCEP)[Results from the first eight questions and the data from Flynn, et al.  are showam (NCEP).First year students did not do as well as physicians at identifying sources of monounsaturated fats. On the other hand, the good discrimination coefficient indicates that knowledge of fat composition is a good indicator of overall knowledge .Although a substantial fraction of cardiologists polled by Flynn knew that carbohydrate raised triglycerides Figure , most intotal amount of fat has decreased, whereas for women there has been a slight increase consistent with the much larger increase in caloric intake among women [prima facie evidence of the effect of macronutrients. Despite the clear correlation between higher carbohydrate, lower fat and obesity, government and health agencies rarely question the appropriateness of the original guidelines and have continued to recommend still higher carbohydrate and still lower fat [Along the same lines, our questionnaire went beyond the area covered by Flynn to consider the changes in diet that have accompanied the epidemic of obesity. It has to be considered very surprising that only 22 % of an educated population knew that the per cent fat in the American diet has decreased Figure ; for menng women . The obsng women  to proviower fat ,30,31. SWe were surprised by some lapses in student knowledge revealed by the questionnaire. The most basic question – which macronutrient is the most energy dense – had only 74 % correct answers. To understand how surprising a response this is, it should be understood that the mean score on exams in this section of the medical course is typically 80 % and, on most exams, several questions are answered correctly by 98–100 % of the class. The National Board of Medical Examiners assumes knowledge of caloric value of macronutrients, and we had expected that it was common knowledge. The questionnaire result indicates that no fact in nutrition is too basic to be excluded from course material.and apple pie for high cholesterol.An overwhelming number of medical students believe that diet is as effective as drugs in lowering total cholesterol. This again attests to the pervasive message that diets control blood cholesterol, an idea continually reinforced by media advertisements, for example, for the cholesterol lowering effect of breakfast cereal. Whereas it is likely that diet is an important influence on CVD, there is, again, the problem of which diet and the question would probably have been better framed, as in questions 2–4, on specific lipid components. It is likely, for example, that many medical students would not know that dietary cholesterol is largely without effect on serum cholesterol. In any case, it is generally acknowledged that, on average, drugs such as statins have a greater impact on cholesterol than currently reported diet interventions. The general effectiveness of statins and the promotion by pharmaceutical companies has, most recently, led to a movement to reinforce the idea that genetics (which can't be controlled by diet) also plays a role. The competing financial interests have produced, in our view, bizarre and unpatriotic (?) television commercials blaming mother Few students in the first year medical class knew that replacing unsaturated fat with carbohydrate was the most damaging substitution in terms of an association with CVD risk. The data from Hu, et al., ,25 shownThese two questions Table  point ouWe recommend that the term complex carbohydrates not be used since, in practice, it has lost its original meaning of polysaccharide. It is interesting that, to some extent, student answers followed the original definition. Most students picked both white bread and whole wheat bred as complex although, with a slight preference for picking whole wheat over white bread as many health professionals and the lay public might.We credit the popular media with the generally good knowledge about the antioxidant vitamins shown in Table These questions were presented as a preview for an upcoming lecture on folate metabolism; therefore it was expected that students would not score very highly Table . We idenMany papers have been written on the need for, and the difficulty in implementing, improvements in teaching nutrition in medical schools ,3,33. SoWith respect to point 1) above, the first year medical school curriculum is undoubtedly very dense in content. Adding new material is difficult, especially if it is of the strictly factual type, e.g. macronutrient composition of particular foods. The "low pressure" quiz used here can, in theory, impart a certain amount of specific knowledge and generate student interest without interrupting the general flow of course work. The quiz provides a venue in which interested students can absorb the information, and become aware of the general area if they need to find the information later. Also, in our view, many subjects taught in basic science courses already have nutritional relevance, e.g., cofactors that come from vitamins, and these ideas should be better emphasized. We point out, when the NAD cofactors are introduced, that one would expect global effects of a deficiency disease because of the number of different enzymes that use these cofactors. Although vitamin deficiencies are rare in the absence of gross malnutrition, the emerging role of hypervitamin therapies  has greaWe see the question of what to teach as the most critical problem in introducing or expanding nutrition education in the medical school course. Individual faculty may be resistant to giving up their own interests, but this may depend on how well the case is made for changing to new topics.The original study by Flynn was designed to test physicians' knowledge and expand it to allow them to better implement ATP III recommendations on serum triglycerides. In combination with other questions that we have introduced, the general problem arises as to whether these recommendations or nutritional data should be taught. We feel that official recommendations, such as ATP III, have some inherent contradictions. Given that low fat diets tend to raise triglycerides, the associated recommendations to reduce dietary fat and to raise carbohydrate intake appear somewhat contradictory. The major focus of ATP III, however, is control of cholesterol but again, the literature is not clear-cut. Thus, whereas the association between cholesterol levels and CVD is generally accepted by all but a minority of critics, the effect of diet, especially reduced fat diets, on CVD, or even cholesterol, is far more controversial. The Chapter on "Diet and Coronary Heart Disease (CHD)" in Willett's Nutritional Epidemiology  is 40 paThe analysis above also bears on the role of low carbohydrate diets in educating students and physicians. We have previously indicated how such diets can be used to teach basic intermediary metabolism  and wherIn summary, what to teach remains very problematic. There are clear inconsistencies in the dietary recommendations of the ATP III and other professional agencies. This has to make one question whether students and physicians should be educated only in currently recommended practice, or whether we should instead emphasize understanding the underlying data. This is especially true, given the disclaimers in the American Heart statement  that "ThA questionnaire, derived in part from one previously published to assess physician knowledge, can be used to determine medical student awareness of nutritional facts. At the same time, such a quiz can be employed as a teaching device to reinforce earlier material, provide preview of new material, or expose students to factual information that is not easily incorporated into a formal course. One of the areas chosen, the effect of macronutrients on obesity and cardiovascular disease, can lead to discussion and focus on important current issues. The performance of first year medical students as well as the performance of the physicians in the previous study suggest that improvement is needed in imparting knowledge about some basic ideas in nutrition. We believe that the focus should be on these ideas rather than on official recommendations with which the ideas are sometimes in conflict.Finally, the questionnaire is intended as a practical method. The authors would be grateful for any information on the outcome of its use and/or any suggestions for improving the quiz itself.HDL: High Density LipoproteinLDL: Low Density LipoproteinTAG: TriacylglycerolTHF: Tetrahydrofolic acidTLC: Therapeutic Lifestyle Changes DietATP III: Third Report of the National Cholesterol Education Program Adult Treatment ProgramNCEP: National Cholesterol Education ProgramGI: glycemic indexGL: glycemic loadCVD: Cardiovascular Disease


Tiny protrusions on the surface of cells might be a more common mechanism for cell communication than previously expected During development, cells need to communicate with each other to establish properly organised and functional tissues. Cells communicate with each other in various ways, such as by secreting and receiving diffusible molecules  or by establishing intercellular connections (gap junctions and cell protrusions) to allow a direct exchange of instructive factors. A recent paper has shown that communication via tiny cell protrusions might be a more common mechanism than previously expected .Drosophila wing and eye imaginal discs (precursors of adult structures) that signals modulating the growth and patterning of one epithelial layer of cells are received through microtubule-based cell extensions arising from the apposing epithelium  on the protrusion could bind and activate receptors displayed on the surface of the receiving cell .The paper by Rustom and colleagues has provided a new outlook on the role of cell protrusions, by reporting a novel mechanism employed to transmit signals between cells connected by a protrusion. Surprisingly, they did not observe any of the mechanisms described above. Rather, transfer of molecules and organelles occurred directly from the cytoplasm of one cell to the other, passing through a protrusion that established membrane continuity between the connected cells.Using rat PC12 cells, Rustom and colleagues observed ultrafine protrusions  connecting sparse cells in culture . SimilarAiming to investigate how TNTs were linking cells, the authors performed scanning and transmission electron microscopy of TNTs. They observed a seamless transition between TNTs and the cells they were connected to, suggesting that indeed there was continuity between the membranes of the two connected cells. Rustom and colleagues then went on to test whether TNTs could be used to transmit signals between cells. The experimental approach used was to mark two populations of cells in a distinct way, either by introducing genes that encoded proteins tagged with green fluorescent proteins or by using dyes. The two different cell populations were then mixed, cocultured, and analysed for transfer of marked proteins or dye-stained organelles from one cell to another, between cells that were differently marked and connected by a TNT. Strikingly, soluble cytoplasmic molecules could not pass freely along the TNTs (with actin tagged with green fluorescent protein being the only exception), whereas membrane-bound proteins were transferred along TNTs and detected in the receiving cells, further supporting the likelihood of membrane continuity between connected cells. Rustom and colleagues also observed transport of vesicles, which seemed to be unidirectional. Finally, in transfer experiments performed at close to 0 °C, where endo-, exo-, and phagocytosis would be blocked, vesicle exchange still occurred, suggesting that these events are not required for vesicle transfer and further supporting the idea that membrane continuity exists between connected cells. By contrast, interfering with actin polymerization, using the drug latrunculin-B, led to protrusion removal and arrest in organelle transfer, indicating that actin is required both for protrusion biogenesis and organelle transport.Taken together, the experiments performed by Rustom and colleagues strongly suggest a role for cell protrusions in cell-to-cell communication. They also provide evidence, in culture, for a novel mechanism used by cell protrusions to transport molecules and organelles. It will be interesting to test whether TNTs also exist in living tissues and, if so, what molecules they transport. TNTs could be distinct from the protrusions known so far and could be responsible for establishing another type of connection between cells. They could connect all cells in a tissue, directly or indirectly, establishing a global interaction network potentially important in exchanging basic survival information as well as positional cues .Another interesting question is how connections such as TNTs are established. Rustom et al. have shown that, initially, many filopodial extensions arise from one cell and are directed toward a neighbour. As soon as one of them reaches the target, it is stabilised, while the others degenerate. It is possible that membrane fusion occurs between the tip of the protrusion and the planar plasma membrane of the target cell. However, membrane fusion can be more easily achieved if the tips of two cell protrusions fuse with each other, thus suggesting the participation, in the process of membrane fusion, of microvilli or other tiny protrusions belonging to the target cell. Fusion between two protrusions is reported to rely on the cylindrical shape and narrow diameter of cell protrusions and also on the localised concentration of adhesion/fusion molecules at the tips of the cell protrusions, such as microvilli, that display particular tip-specific membrane microdomains . The worIn my work as a graduate student, I am trying to address this question. We need to identify the types of cell protrusions that are present in tissues and the molecular complexes localizing on them as well as their functions. To then prove that cell protrusions are important in cell-to-cell communication in tissues, we would need to remove the protrusions and see how this affects tissue architecture and function. However, the necessary tools are still missing, given the lack of knowledge of the specific molecules important for the biogenesis of these protrusions. Thus far, the function of cell protrusions has been hypothesized mainly on the basis of their location in tissues and on crude attempts to remove them, for example by altering the actin cytoskeleton or even by removing the entire epithelium they belong to.The paper by Rustom et al. has shed some new light on these still mysterious cellular arms and has further boosted my interest in this emerging field of cell and developmental biology.


The use of complementary and alternative products including Biological Based Therapy (BBT) has increased among patients with various medical illnesses and conditions. The studies assessing the prevalence of BBT use among patients with cardiovascular diseases are limited. Therefore, an evaluation of BBT in this patient population would be beneficial. This was a survey designed to determine the effects of demographics on the use of Biological Based Therapy (BBT) in patients with cardiovascular diseases. The objective of this study was to determine the effect of the education level on the use of BBT in cardiovascular patients. This survey also assessed the perceptions of users regarding the safety/efficacy of BBT, types of BBT used and potential BBT-drug interactions.The survey instrument was designed to assess the findings. Patients were interviewed from February 2001 to December 2002. 198 inpatients with cardiovascular diseases (94 BBT users and 104 non-users) in a university hospital were included in the study.Users had a significantly higher level of education than non-users . Top 10 BBT products used were vitamin E [41(43.6%)], vitamin C [30(31.9%)], multivitamins [24(25.5%)], calcium [19(20.2%)], vitamin B complex [17(18.1%)], fish oil [12(12.8%)], coenzyme Q10 [11(11.7%)], glucosamine [10(10.6%)], magnesium [8(8.5%)] and vitamin D [6(6.4%)]. Sixty percent of users' physicians knew of the BBT use. Compared to non-users, users believed BBT to be safer (p < 0.001) and more effective (p < 0.001) than prescription drugs. Forty-two potential drug-BBT interactions were identified.Incidence of use of BBT in cardiovascular patients is high (47.5%), as is the risk of potential drug interaction. Health care providers need to monitor BBT use in patients with cardiovascular diseases. The use of complementary and alternative medicine (CAM), defined by the United States National Center for CAM as a group of diverse medical and health care systems, practices, and products that are not presently considered to be a part of conventional medicine, has grown tremendously in the United States -4. A recSimilar to other chronic medical conditions, patients suffering from a variety of cardiovascular diseases including coronary heart disease, congestive heart failure, stroke, arrhythmia and congenital cardiovascular defects, may also be looking to CAM to prevent or treat their illnesses. This is particularly likely since a number of BBT products including vitamin E, vitamin C, beta-carotene, fish oils, and coenzyme Q10 have been evaluated for prevention and/or treatment of cardiovascular diseases -25.Despite a wide array of available BBT for cardiovascular conditions, studies evaluating the prevalence of usage of these agents are limited -25. As CThe primary objective of this study was to determine the effect of the education level on the use of BBT in cardiovascular patients. The study also investigated the attitudes and beliefs towards BBT by patients with cardiovascular diseases. In addition, patient perceptions regarding the safety and efficacy of BBT, common BBT used and a list of potential BBT-drug interactions were reviewed.This is a cross-sectional, descriptive study utilizing structured interviews to assess the level of education, usage, beliefs and attitudes towards BBT among inpatients with cardiovascular diseases. The study was conducted in the Cardiac Care Center at the Mount Sinai Hospital, New York, USA from February 2001 to December 2002. Participants provided informed consent and were interviewed by one of the investigators. To maintain consistency of the interview and to prevent interviewer bias, a scripted letter was drafted for the investigators to invite the patients to participate in the study and to explain the process of the study. After a patient was enrolled, an investigator read the survey questions verbatim to the patient and tried not to elaborate whenever possible. Patients were included in the study if they had at least one of the following diagnoses: cardiovascular disease(s) including congestive heart failure, coronary heart disease, thromboembolic diseases, valvular heart disease, arrhythmia, vascular aneurysm, peripheral vascular disease, pulmonary hypertension, congenital heart disease and post heart transplant. Additional inclusion criteria included being 18 years of age or older, English speaking, no documented cognitive deficits precluding the patient from understanding the interviewer, and willingness to provide an informed consent. Prior to patient contact, the attending physicians of the eligible patients were contacted and informed about the study. If the attending physicians chose not to have their patients participate in the study, the patients were not included. Upon agreement of the physicians, subjects were invited to participate and were asked to sign an informed consent at their convenience prior to being interviewed. This study was approved by the Institutional Review Boards of Mount Sinai Hospital and Long Island University.® Database ) as compared with nonusers, . In contrast, 42 (40%) nonusers finished high school versus 23 (24%) of users, p = 0.023. There were no significant differences between users and nonusers in other demographic variables.The distribution of cardiovascular and noncardiovascular diseases reported by the sample is presented in Table The mean number of traditional prescription and nonprescription medications taken by the users of BBT (7 [range 1–15]) and nonusers (7 [range 0–18]) was not significantly different, p = 0.445. The most common cardiovascular medications prescribed for both users and nonusers were diuretics , followed by aspirin  and beta-blockers . The cardiovascular and noncardiovascular medications taken by both groups were not different.The BBT products utilized by cardiovascular patients are presented in Table The patients reported that their physicians were aware of their BBT use in 60% of the instances and pharmacists were aware in 32% of the cases. Only 33% of users reported that they were asked about BBT use during a history/physical examination by a health care professional. The patients were not surveyed about their pharmacists' assessments of BBT use.A greater percentage of users (74.5%) of BBT reported that they believed these products to be safe substances as compared with nonusers (26.2%), p < 0.001. Likewise, significantly more users believed that BBT was effective (70.2%) compared to nonusers (30.1%), p < 0.001. More nonusers (72%) than users (45%) did not know whether BBT products work better, as well as, or worse than traditional medications, p < 0.001. More users than nonusers believed that BBT works as well as traditional medications . Concerning adverse effects, more users (44.7%) of BBT than nonusers (17.5%) believed that BBT causes fewer side effects than traditional medications, p < 0.001. At the same time, 32.2% of users and 62.1% of nonusers did not know whether BBT causes more or fewer side effects than traditional prescription medications, p < 0.001.Examination of patients' prescription and nonprescription medication profiles and BBT utilized, revealed 42 potential drug-BBT interactions. The onset of the interaction and the degree of severity were classified according to that used by the MicroMedex HealthCare Series Integraded Index  and the Despite a wide array of available BBT for cardiovascular conditions, studies evaluating the prevalence of their usage are limited. A review of the literature at the time of study initiation (January 2001) and more recently (June 2004) identified five studies in this area, and only a few included a broad spectrum of cardiovascular disease patients -25.Wood et al. conducted a telephone survey in 107 patients randomly selected from the "Improving Cardiovascular Outcomes in Nova Scotia" database . A majorIn our study, the lifetime prevalence of BBT use in the sample of 198 cardiovascular patients was 47.5%, which is very similar to the results reported by Eisenberg and colleagues in the general population (42%) . CompariSimilar to Ackman's study , the mosSimilar to other studies ,3,6,21, Among the demographic variables collected in this analysis, no parameter other than the level of education had a significant impact on whether patients used BBT. The present investigation revealed that the education level among users (63% received some college or college and graduate school education) of BBT was higher than that of the nonusers (32%), p < 0.001. This finding has also been reported in other studies [The majority of BBT users (as compared to nonusers) in the current investigation believed that BBT products are safe, effective and cause fewer side effects as compared to prescription medications. These results are consistent with the findings by other investigators ,21, suppSeveral limitations of the study need to be addressed. Those that are intrinsically related to survey data collection in general include potential bias of responders and nonresponders. Since participation in this study was voluntary, patients who chose to participate in the study may be more motivated and knowledgeable about the subject of BBT as compared to those who refused to participate. Therefore, the results of this study may not be applicable to the entire cardiovascular patient population. Also, a cross-sectional nature of this study precludes drawing any definitive conclusions regarding cause and effect relationships. For example, one cannot definitively conclude that more education will significantly effect one's decision to take BBT because other factors such as exposure to these types of products may have influenced more educated consumers to take these products.As with other surveys, recall bias cannot be eliminated as data collection relied on patients' self-report, rather than objectively documenting BBT use. Additionally, with surveys there is always a possibility of misunderstanding the questions and responses, miscommunication between the interviewer and the patients, and inaccurate recording of the information. The majority of the survey collected factual information . However, the questions that evaluated patients' perception on the safety and efficacy of conventional medicine and BBT were not validated.This study was conducted in a large urban inner city hospital; the results may not be extrapolated to other cities or clinical settings. Another limitation may be the exclusion of non-English speaking patients, which may be particularly important since there are certain ethnic groups who are documented to use more BBT than are other ethnic groups . Finally, terminology varies greatly in the published literature. Some authors use the term CAM in all instances, others differentiate CAM from BBT, etc. As a result, it is very difficult to compare published reports with certainty.In recent years, the interest of using BBT in disease management has increased dramatically in the medical and layman communities. The amount of valid scientific research in this area of therapy continues to increase. Yet, there are still many unknowns concerning BBT, especially in the area of adverse effects and drug interactions. The finding of a high prevalence of BBT (47.5%) use among cardiovascular patients and the lack of communication between patients and their physicians/pharmacists should be addressed by the health care community. Higher education level, as shown in the present study and other previous investigations ,3,6,22, The author(s) declare that they have no competing interests.LC participated in literature review, protocol development, collected and analyzed data, and submitted the manuscript for publication. DB collected the data. JWMC participated in study design, data analysis and preparation of manuscript. VR participated in design development and data analysis. HLK proposed the project and reviewed the manuscript. GCC, JM and BM participated in protocol implementation and manuscript review. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:Appendix A – Biological based therapy survey, It's a survey tool utilized in the study to collect patient data.Click here for file


The mitogen-activated protein kinases (MAPKs) have been shown to participate in a wide array of cellular functions. A role for some MAPKs  has been documented in response to certain physiological stimuli, such as ischemia, visceral pain and electroconvulsive shock. We recently demonstrated that restraint stress activates the Erk MAPK pathway, but not c-Jun-N-terminal kinase/stress-activated protein kinase (JNK/SAPK) or p38MAPK, in several rat brain regions. In the present study, we investigated the effects of a different stressor, acute forced swim stress, on the phosphorylation (P) state of these MAPKs in the hippocampus, neocortex, prefrontal cortex, amygdala and striatum. In addition, effects on the phosphorylation state of the upstream activators of the MAPKs, their respective MAPK kinases , were determined. Finally, because the Erk pathway can activate c-AMP response element (CRE) binding (CREB) protein, and swim stress has recently been reported to enhance CREB phosphorylation, changes in P-CREB were also examined.A single 15 min session of forced swimming increased P-Erk2 levels 2–3-fold in the neocortex, prefrontal cortex and striatum, but not in the hippocampus or amygdala. P-JNK levels (P-JNK1 and/or P-JNK2/3) were increased in all brain regions about 2–5-fold, whereas P-p38MAPK levels remained essentially unchanged. Surprisingly, levels of the phosphorylated MAPKKs, P-MEK1/2 and P-MKK4  were increased in all five brain regions, and much more dramatically . Consistent with the lack of forced swim on phosphorylation of p38MAPK, there appeared to be no change in levels of its activator, P-MKK3/6. P-CREB was increased in all but cortical  areas.Swim stress specifically and markedly enhanced the phosphorylation of the MAPKKs P-MEK1/2 and P-MKK4 in all brain regions tested without apparent alteration in the phosphorylation of P-MKK3/6. Curiously, phosphorylation of their cognate substrates (Erk and JNK) was increased to a much more modest extent, and in some brain regions was not altered. Similarly, there was a region-specific discrepancy between Erk and CREB phosphorylation. Possible explanations for these findings and comparison with the effects of restraint stress will be discussed. Thus, activation of the Erk pathway via both a cAMP/PKA-dependent mechanism leading to activation of CREB, and a non-cyclase-linked pathway, leading to activation of CREB and/or the serum response element (SRE) binding protein Elk-1, have been shown to occur in the hippocampus during acquisition of learning, memory consolidation and long-term potentiation (LTP) [Increasing evidence implicates stress as an important factor in the vulnerability to depressive and other mental illnesses -3. Conseon (LTP) -8. It ison (LTP) .The term MAPK is used to denote a family of signal transduction mediators, extensively distributed throughout the central nervous system ,11, thatc-fos gene and/or Fos protein expression in many brain regions) [c-fos and zif286. P-CREB and P-Elk-1, by binding to these elements, enhance the transcription of the IEGs and regulate gene expression [c-fos expression, and on the other, that certain stressful stimuli (see above) activated Erk, it was reasonable to predict that acute physiological stressors would activate the Erk (and perhaps other) MAPK pathway(s). The brain regions most likely to show such an effect would be those implicated in the response to stress, including the hippocampus [Subjecting rats or mice to acute stressors such as restraint or forced swim results in activation of regions) -24. CRE pression ,26. Recepression . Thus, gpocampus , prefronpocampus -32 and opocampus ,33, and pocampus ,34-36. Ipocampus . The prepocampus .An acute 15 min session of forced swimming produced obvious large increases in the phosphorylation of the MAPKK MEK1/2 in the hippocampus, neocortex, prefrontal cortex, amygdala and striatum  in the hippocampus  may account for the difference in results. Note, moreover, that the regional pattern of changes in P-CREB , except for the increase in P-MEK1/2 in neocortex, which peaked at 5 min Fig. . The levThe results presented establish unambiguously that forced swim stress elicits a rapid and profound but relatively transient increase in the phosphorylation (and consequently the presumed activation) of the MEK-Erk and MKK4-JNK signaling pathways in, at the least, the five brain regions examined. These effects displayed specificity in that the MKK3/6-p38MAPK pathway was essentially unchanged.  and the corresponding MAPKs (P-Erk and P-JNK). It is obvious from Figs. Nevertheless, differences in phosphorylation stoichiometry cannot explain why, in some brain regions  increases in P-Erk levels in hippocampus and prefrontal and cingulate cortex . As for c-fos gene expression) [via NMDA glutamate receptor-stimulated calcium influx [The nature of the extracellular signaling molecule(s) that are recruited upon subjecting animals to stress are unknown. The pathways leading from stimulation of a particular receptor on the cell surface to activation of MAPK signaling cascades are diverse and complex ,14,47-50ression) -24, and m influx -56. Strem influx , which mm influx . Thus, im influx ). NMDA rm influx -60. Intem influx . In prelAcute swim stress initiated a rapid increase in phosphorylation of the MAPKKs MEK1/2 and MKK4 in hippocampus, neocortex, prefrontal cortex, amygdala and striatum. Concomitantly, their corresponding substrates Erk and JNK were also phosphorylated, but not always in register with the changes in the MAPKKs. Moreover, the magnitude of the increase in phosphorylation of MEK1/2 and MKK4 was much greater than for their cognate substrates. While it is clear that stressors such as forced swim and restraint activate these signaling pathways, much work will be required to define the mechanism(s) responsible for these effects, and to relate potential alterations in the activity of these molecules to stress-related influences on the development of anxiety and depressive disorders.Guide for the Care and Use of Laboratory Animals, and were approved by the NYU School of Medicine Institutional Animal Care and Use Committee.Male Wistar rats  were used throughout. Rats were housed 2/cage and maintained on a 12 h light/dark cycle with food and water ad libitum; they were acclimated to handling and transportation for 2–3 weeks before being used in experiments. All experimental procedures were carried out in accordance with the NIH ® protease inhibitor cocktail was from Roche Molecular Biochemicals, Indianapolis, IN and colored molecular weight markers were purchased from Bio-Rad, Hercules, CA. Biotinylated protein ladder marker and anti-biotin were from Cell Signaling Technology (CST), Beverly, MA. West Pico Super Signal enhanced chemiluminescence (ECL) reagent was obtained from Pierce Biotechnology, Rockford, IL, and Re-Blot Plus stripping buffer was from Chemicon International, Temecula, CA. All primary antibodies were rabbit polyclonal except as noted. Because suppliers often offer more than a single antibody for the same antigen, catalog numbers are specified in parentheses. P-Erk , pan Erk (sc-93) and pan MKK4/SEK1 (sc-964) were all from Santa Cruz Biotechnologies, Santa Cruz, CA. The following antibodies were obtained from CST: P-MEK1/2 (9121), pan MEK1/2 (9122), P-MKK4/SEK1 (9151), P-JNK , pan JNK (9252), P-MKK3/6 (9231), pan CREB (9192) and pan p38MAPK (9212). P-CREB (06-519) was from Upstate Biotechnology, Inc. , while P-p38 was either from CST (9212) or Chemicon (AB3828). Control cell extracts for P-MKK3/6 were obtained from CST: +/- UV-treated NIH/3T3 cell lysates (9233) and +/- anisomycin-treated C6 glioma cell lysates (9213).Precast 10% polyacrylamide gels were obtained from Cambrex BioScience, Rockland, ME. CompleteGroups of rats (n = 4–6) were subjected to forced swim stress in a round glass tank (24 cm W × 44 cm H) filled to a depth of 30 cm with water (25 ± 1°C). In most experiments rats were forced to swim for 15 min and sacrificed by decapitation immediately after narcotization with carbon dioxide for 20 sec. In some experiments the duration of swim stress (2–15 min) or the post-swim interval (0–60 min) was varied as described in the legends to Figures. Control rats were not treated and were sacrificed in parallel as above.Brains were rapidly removed, briefly chilled in saline and placed in a stainless steel brain matrix . Sections of 1–2 mm in thickness were cut, frozen quickly on dry ice, and brain areas of interest  were micropunched using the atlas of Paxinos and Watson  as a gui3VO4, 1 mM NaF, 0.5 μM okadaic acid and Complete® protease inhibitor cocktail). After centrifugation at 30,000 g for 20 min, lysates were mixed with 5X sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min and stored at -80°C. Protein content of lysates was determined using the bicinchoninic acid assay kit (Pierce).Tissues were mechanically disrupted, in glass homogenizers using a Teflon pestle, in 10–20 volumes of buffer  were separated by SDS-PAGE on precast polyacrylamide gels. Gels were also loaded with colored molecular weight markers to assess electrophoretic transfer, and biotinylated protein ladder marker to estimate molecular weights of bands of interest. Following electrophoretic transfer to nitrocellulose, blots were incubated in blocking buffer ) for 1 hr at room temperature (RT), washed 3 × 10 min in TBST and incubated with primary phospho-specific antibodies overnight at 4°C . Blots were washed 3× in TBST, incubated with the appropriate secondary antibody plus anti-biotin for 1 hr at RT, washed again, treated with ECL reagent and exposed to film. Blots were then stripped by incubation for 15 min at RT with Re-Blot Plus, re-blocked, washed and incubated for 1 hr at RT with the corresponding pan antibody which recognizes total antigen protein (phosphorylated and nonphosphorylated). This was followed by incubation for 1 hr at RT with the appropriate secondary antibody plus anti-biotin. Antigens were again visualized by treatment with ECL reagent and exposure to film.A computerized image analysis system  was utilized to analyze immunoblots. Image analysis was carried out as described previously . For eact test .Control and swim-stressed groups were compared using Student's C-PS conducted the majority of the immunoblotting studies. YT performed some of the experiments and quantitated many of the films using image analysis. CS assisted in the design of some of the experiments, carried out the MKK3/6 experiments, and assisted in the writing of the manuscript. EM designed the study, supervised and facilitated the experiments, analyzed the data and wrote the manuscript.


You are what you eat and drink. Steak can sit in your stomach or orange juice wind through your intestines, but they only become part of your body once they're taken up by your cells. First, foods must be reduced to a soup of proteins, fats, sugars, and so on. But even then, getting these materials into a cell isn't as simple as sticking them in your mouth. For one, there's the membrane enclosing a cell. Simply puncturing a hole in the membrane would spill the cell's contents, harming or killing the cell.Instead, all eukaryotes—organisms whose cells have nuclei—use a carefully orchestrated process called endocytosis to bring materials into their cells. Eukaryotic cells first form cavities in their cell membrane that surround nearby particles or fluid. These pockets seal shut and bud off into the cell to form small membrane-bound sacs called vesicles.When taking in fluids, eukaryotic cells use two distinct mechanisms—to take tiny sips or huge gulps. With one process, called pinocytosis, cells continually form small pockets in the cell membrane that enclose small droplets of fluid in vesicles called pinosomes. These newly formed vesicles, called early endosomes, bud off from the membrane and fuse with other early endosomes. In one form of pinocytosis, the vesicles are encaged by a protein called clathrin that tightly constrains their size. These carriers incorporate membrane constituents  with very high selectivity. In macropinocytosis, on the other hand, large ruffles in the membrane engulf mass quantities of fluid in vesicles known as macropinosomes.Beyond taking in nutrients, these processes are essential to the function of many organs—from the brain, where nerve cells receive other cells' chemical signals by pinocytosis, to the kidney, where cells use macropinocytosis to take in waste fluids for processing. Macropinocytosis is also relevant to cancer cells; it has long been known that oncogenes dramatically induce this endocytic process, affecting the signaling status of these cells. But compared with other types of endocytosis, molecular biologists know surprisingly little of the mechanisms behind macropinocytosis. They do know that the Rab5 protein—an enzyme that coordinates a complex network of other proteins, called effectors—is crucial for both pinocytosis and macropinocytosis.PLoS Biology, Marino Zerial and colleagues have found a new protein, which they named Rabankyrin-5, that forms a further link between these two mechanisms for fluid uptake. The protein is necessary for macropinocytosis, and its levels control the rate of this process. In addition, Rabankyrin-5 helps regulate endosome trafficking and coordinates this mechanism with macropinocytosis.Now, as reported in this issue of In two commonly used human and mouse cell lines, the researchers found the protein Rabankyrin-5 along with Rab5 on both types of pinosomes, early endosomes and macropinosomes. The early endosomes usually fuse with one another inside the cell, but when the researchers blocked Rabankyrin-5 activity, this fusion fell sharply. Suppressing Rabankyrin-5 activity also stifled macropinocytosis; overexpressing the effector, on the other hand, sent macropinocytosis into overdrive.The researchers also looked at endocytosis in mouse kidney and canine kidney cell lines. Inside the kidney, fluid-carrying ducts are lined with epithelial cells that take up liquids through their exposed surface. The researchers found Rabankyrin-5 predominately on vesicles at this surface, and as in the other experiments, overexpression of the protein promoted macropinocytosis. Together, these findings suggest Rabankyrin-5 plays a role in regulating this form of fluid uptake and plays a role in kidney function. The discovery of Rabankyrin-5 involvement in macropinocytosis also has implications for other physiological and pathological mechanisms such as the immune system response, defense against pathogens, and hyperactivation of signaling pathways in cancer cells.Rabankyrin-5 contains various regions that bind other proteins and also lipids found in cell membranes, suggesting the protein plays a mechanical role in forming vesicles. The protein also has regions found on other proteins that are involved in signaling and development, so it may help direct vesicles' traffic within the cell. The protein also has regions characteristic of proteins involved in clathrin-dependent endocytosis, which fits with the researchers' finding that Rabankyrin-5 affects pinocytosis.All told, Rabankyrin-5 appears to form a bridge between two distinct mechanisms, pinocytosis and macropinocytosis, that cells use to take in fluids. While the details of how Rabankyrin-5 functions are still unclear, these findings give researchers a new handle for grasping how macropinocytosis works and how cells control when and how much they drink in their surroundings.


Wireless local area networks (WLANs) are considered the next generation of clinical data network. They open the possibility for capturing clinical data in a prehospital setting  using various devices, such as personal digital assistants, laptops, digital electrocardiogram (EKG) machines, and even cellular phones, and transmitting the captured data to a physician or hospital. The transmission rate is crucial to the applicability of the technology in the prehospital setting.We created two separate WLANs to simulate a virtual local are network environment such as in a patient's home or an emergency room (ER). The effects of different methods of data transmission, number of clients, and roaming among different access points on the file transfer rate were determined.The present results suggest that it is feasible to transfer small files such as patient demographics and EKG data from the patient's home to the ER at a reasonable speed. Encryption, user control, and access control were implemented and results discussed.Implementing a WLAN in a centrally managed and multiple-layer-controlled access control server is the key to ensuring its security and accessibility. Future studies should focus on product capacity, speed, compatibility, interoperability, and security management. The development of the Internet has encouraged doctors to use computers and hospitals to use wireless communications , since wAt present, patient data such as electrocardiograms (EKG) and demographics are seldom sent from the prehospital environment to the ER before the ambulance arrives . As a reThe objective of this study was to assess the ability of wireless technology to facilitate data communication between a prehospital setting and an ER . The idea was for all the data collected by paramedical personnel to be transmitted to an ER server from the patient's home, on the way to the ER, or upon arrival at the ER. Thus the transmission rate is crucial to the usefulness and applicability of the technology. To test the feasibility of wireless data transmission under the various scenarios, two separate WLANs were created, one around our office and another in a house. In this report, we discuss our testing of the wireless technology, and its potentials and limitations in simulated prehospital settings.The WLAN products used (Aironet 340 and 350 series wireless client adaptors and access points (APs); Cisco) offered 11-megabits-per-second (Mbps) transmission rates, built-in security features (including 40- to 128-bit encryption) and Web-based management. The transmission rates of files of different sizes were measured with different APs, patch antennae, clients, and transfer methods. The security of patient data was ensured using a centrally managed Access Control Server (ACS). Other issues such as standards, roaming, and cost are also discussed here.Within a WLAN, data are transmitted between a server and its wireless clients via an AP antenna. Both workstations and laptops were used here as servers, and file transfer rates were measured for both systems. We used Gateway Select series, Dell Inspiron series, and a Toshiba Satellite laptop computer as clients. The computers had CPU operating at 0.8–1.2 GHz, 256–1024 MB of RAM, and 10–40 GB hard drives, and all ran the Microsoft Windows 2000 Professional operating system. PDAs  were also tested as wireless clients. The wireless coverage was tested using two APs  and a patch antenna . The feasibility of using cellular phones  in data transmission in the area not covered by the WLAN was also tested.The various software used in the study for wireless client management, file transfer, and access control included the Aironet Client Utility (Cisco), Link Status Meter , the ACS , Phone Tools (BVRP Software) for faxing, and file transfer protocol (FTP) for measuring the file transmission rate. The software LSM classifies the link status as the percentage of maximum signal strength and quality: "excellent" (>75%), "good" (40–75%), "fair" (20–40%), or "poor" (<20%); where signal strength and quality refer to the client adapter's radio signal at the time packets are being received, quantified as bytes transmitted and received and the errors that occur. Detailed descriptions of the mentioned software are available from the manuals provided by the vendors.The Aironet 340 and 350 series APs were tested by a two-step approach. In the first step, one AP was connected directly with the server that was not connected to the campus Ethernet backbone. In the second step, the AP was assigned a public Internet Protocol (IP) address and connected to the Ethernet backbone in the Susan Mott Webb Nutrition (Webb) Building at the University of Alabama at Birmingham. The IP address was assigned to an AP through either HyperTerminal or a Web console using a Web browser. An administrator ID and password were then created to enhance the Web console security. The client computer required a type II PCMCIA  card slot. Every client needed a functional IP address to become associated with the AP. The Wired Equivalent Privacy (WEP) keys were enabled for both the AP and the clients to ensure two-way authentication.The coverage of the WLAN was found to highly structure dependent. The floor of the Webb building measures about 60 by 25 meters. A single Aironet 340 AP was unable to cover the entire floor with a "good" link status. This was achieved using two (more powerful) Aironet 350 APs. Achieving the "excellent" link status on the floor required the use of the S2406P (Cushcraft) patch antenna. The wireless clients associated with the AP had a "fair" or "good" status one floor up and one floor down from the floor where the AP was located. There was a small area outside the 5-floor building in which the clients could associate with the AP with a "poor" status.In order to test the feasibility of implementing an isolated LAN around a patient's home, we chose a two-story house with the layout shown in Figure To quantify the baseline signal strength, one Aironet 350 AP was tested in the open area: "excellent", "good", "fair", and "poor" link statuses were obtained within 10, 25, 50, and 100 feet , respectively. As shown in Table The different methods and directions of file transfer might affect the rate. Methods such as FTP transfer, copying and pasting between folders, and Microsoft Briefcase synchronization were tested. Other factors that may affect transmission were also tested, including the initiation direction  and relative physical motion. One Gateway workstation and two Inspiron 4000 laptops were used. To simplify the test, a single medium-sized file (50 MB) was chosen for the purpose. We chose a 50-MB file since it corresponds to a typical high-quality EKG image. As indicated in Table We chose Ipaq Pocket PCs as PDAs due to their relatively large amount of RAM compared to other PDAs, and a cellular phone (Motorola) as alternative data transmission device when the PDA was outside the WLAN coverage , is not available). The transfer rates for single files of different sizes are summarized in Table A Cisco V3 ACS was used to improve the security of the network . As showWhen managing a WLAN with many clients, there are often insufficient public IP addresses. The solution is to use either the Dynamic Host Configuration Protocol (DHCP) or a private LAN with a Network Address Translation (NAT) server. DHCP services are not easily managed and sometimes create security challenges to network administrators in determining the user's identity. A private LAN seems to be a better solution and more applicable in most ER environments where many wireless clients may transmit at same times, thus maintaining relatively high-speed connections. Figure Health-care organizations have traditionally been slow in accepting WLAN technology in clinical practice. One of the major concerns has been its cost ,16, follAfter collecting all patient data at a patient's home, the data must ultimately be transferred to the ER. This involves two critical synchronization steps: (1) from the patient's home to a server on the ambulance, and (2) from the ambulance to the ER . Here the WLAN was employed for both of these steps, using Microsoft Briefcase and Windows Workgroups. Automatic synchronization with the destination server and batch synchronization were desired. The ultimate objective is, however, to link the two synchronization steps using a long-range antenna that reaches up to 25 miles . This would significantly shorten the time needed to transfer data from a patient's home to the ER, since the data will reach a ER WLAN earlier. We are currently performing the associated experiments. We also tested the use of a cellular phone and other types of PDA  with network capabilities in transmitting a small (up to 50 MB) but critical file. The results suggested that cellular phones or PDAs with network cards can be effective alternatives to the use of a long-range antenna to transmit data from a patient's home to the ER.EKG data are considered very valuable in the early detection, early intervention, and possibly better outcome of heart attack patients . The useThe WLAN operates at 2.42 GHz with an output power of 100 mW, which may pose a risk of interference with medical devices using similar frequencies. Previous studies have shown that a WLAN may interfere with medical devices in close proximity  but is uThe major concern over a WLAN is its security -20, espeThe IEEE 802.11 specification addresses both the physical and MAC layers , and the OpenAir 2.4 interface standard is derived from the Wireless LAN Interoperability Forum  and needThe present study was mainly based on the Windows operating system and Cisco wireless products. IEEE 802.11a products for the next generation of WLANs are emerging quickly from various vendors. Hence the stability, compatibility, and interoperability with other vendors require further evaluation. Although currently it is relatively expensive to implement a WLAN using this new protocol, the prices and capabilities are expected to improve within the near future.Application of WLAN technology will help both paramedics and other health-care professionals in their daily acquisition of information in a localized area such as within a patient's home, an office, a small clinic, or an ER. Implementing a WLAN in a centrally managed and multiple-layer-controlled ACS is the key to ensuring its security and accessibility. Future studies should focus on product capacity, speed, compatibility, interoperability, and security management.None declared.D. Chen, the principal investigator, was most involved in conducting the experiments. H.F. Orthner participated in data collection in the simulated patient's home. H.F. Orthner was the sponsor and S.-J. Soong and G.J. Grimes were the advisors of the fellowship awarded to D. Chen from the National Library of Medicine, National Institute of Health, and they contributed significantly to the design, coordination, and performing of experiments.The pre-publication history for this paper can be accessed here:


We report that mon encodes the zebrafish ortholog of mammalian transcriptional intermediary factor 1γ (TIF1γ) (or TRIM33), a member of the TIF1 family of coactivators and corepressors. During development, hematopoietic progenitor cells in mon mutants fail to express normal levels of hematopoietic transcription factors, including gata1, and undergo apoptosis. Three different mon mutant alleles each encode premature stop codons, and enforced expression of wild-type tif1γ mRNA rescues embryonic hematopoiesis in homozygous mon mutants. Surprisingly, a high level of zygotic tif1γ mRNA expression delineates ventral mesoderm during hematopoietic stem cell and progenitor formation prior to gata1 expression. Transplantation studies reveal that tif1γ functions in a cell-autonomous manner during the differentiation of erythroid precursors. Studies in murine erythroid cell lines demonstrate that Tif1γ protein is localized within novel nuclear foci, and expression decreases during erythroid cell maturation. Our results establish a major role for this transcriptional intermediary factor in the differentiation of hematopoietic cells in vertebrates.Hematopoiesis is precisely orchestrated by lineage-specific DNA-binding proteins that regulate transcription in concert with coactivators and corepressors. Mutations in the zebrafish  A new gene acting early in red blood cell development is discovered by genetic analysis in zebrafish. The gene encodes a member of a well-known family of transcription factors Hematopoiesis involves the coordinated processes of cell proliferation and differentiation of a relatively small number of progenitor cells into billions of circulating red and white blood cells . Hematopgata1 normally in embryonic hematopoietic precursors (moonshine (mon), and another group named a noncomplementing allele vampire , a member of the TIF1 family of transcriptional coactivators and corepressors  visible in circulation ; however, based on TUNEL staining, the differentiating erythroid cells undergo programmed cell death from the 12-somite stage to 22 h postfertilization (hpf)  of all tested alleles survive to adulthood. Adult mon mutants show cardiac hypertrophy, presumably due to the severe anemia leading to a high output state /WIK strain hybrid parents carrying the tg234mon allele. The mon mutant gene was positioned on Chromosome 8 between microsatellite markers z987 and z11001 (http://www.ensembl.org/Danio_rerio/) containing a predicted zebrafish TIF1 family gene. This PAC was hybridized to a kidney cDNA library, resulting in the isolation of four clones that represented the same gene.We identified the d z11001 A . Therefore, the hypomorphic larval phenotype of the m262mon allele is likely due to partial loss of mon function or expression. The presence of mutations in each of the mon alleles indicates that defective Tif1γ function is the cause of the mon phenotype.We have identified ethyl-nitrosourea (ENU)-induced point mutations in three alleles of mon C and 3D,tif1γ is expressed in hematopoietic mesoderm, we next examined zebrafish embryos by whole-mount in situ hybridization   . This grd island . The tifse Tif1γ A and 4B.tif1γ. Using zebrafish expressed sequence tag (EST) sequences, we designed primers to RT-PCR amplify a TIF1-related cDNA from embryonic 10-hpf and 24-hpf RNA. This cDNA encodes a predicted zebrafish ortholog of human TIF1α based on predicted amino acid sequences  of tg234mon mutants show significant  rescue of circulating hemoglobinized RBCs in comparison to control sibling mutants (n = 75). Based on the correction of the jagged fin-fold phenotype (mon did not result in expanded blood cell numbers in wild-type embryos and was not toxic at doses that rescue the phenotype of mon mutants (unpublished data). Since there were no expanded or ectopic blood populations in the embryos, these rescue experiments suggest that mon functions as a permissive factor required for hematopoiesis.To further confirm that a mutation in the zebrafish eriments . Microinhenotype , the mestif1γ expression in erythroid cells suggest that it functions as a cell-autonomous regulator of gene expression in hematopoietic cells. In order to test this hypothesis, we transplanted wild-type adult zebrafish kidney marrow cells carrying a gata1:green fluorescent protein (GFP) transgene into 48-hpf mon mutant embryos  of these demonstrated a marked increase in erythroid cells with 100–1,000 GFP+ cells in circulation 6 d later. By day 10, these transplanted embryos showed approximately 3,000 cells in circulation, similar to the number of blood cells in normal embryos. Despite robust reconstitution of blood cells, mutant recipients did not inflate their swim bladders and thus failed to survive longer than nontransplanted sibling controls, all dying by 3 wk of age. In contrast, 13/35 (37%) heterozygous tg234mon transplants survived to early adulthood. Similar transplants of wild-type cells can fully rescue vlad tepes (gata1) mutants . In two different murine erythroleukemia cell lines (MEL and G1E), Tif1γ is also expressed in nuclear foci, and even though the overall Tif1γ protein level is reduced, this nuclear foci localization does not change with differentiation (unpublished data). This provides further support for the hypothesis that Tif1γ acts within novel nuclear foci, during erythroid differentiation.In order to examine the subcellular distribution of Tif1γ protein, we generated an affinity-purified rabbit polyclonal antiserum directed against the C-terminal 15 amino acids conserved in human TIF1γ and mouse Tif1γ. Immunofluorescence of mouse embryo fibroblast nuclei with the anti-Tif1γ antiserum demonstrates that Tif1γ is localized in small nuclear foci A. The lomoonshine  mutant embryos . We also tested for a direct interaction between Tif1γ and Gata1 proteins by coimmunoprecipitation and yeast two-hybrid assays and found no association (unpublished data). Although the mutations in each of these genes arrest cells at a similar stage of development, our results suggest that gata1 and tif1γ act independently. This does not rule out the possibility that parallel genetic pathways involving gata1 and tif1γ, operating together, regulate gene transcription within blood cells.The hematopoietic phenotype of  embryos . In an etif1γ is required as a permissive cofactor for the erythroid lineage-specific control of hematopoietic gene expression. We reasonably predict that Tif1γ protein functions as a transcriptional intermediary factor in hematopoietic progenitor cells given that both TIF1α  for GFP+ cells over a span of 12 d. On day 13 posttransplant, all surviving larvae  were placed in tanks and monitored for survival.Whole kidney marrow cells were isolated from adult Antisera against the human C-terminal TIF1γ sequence RRKRLKSDERPVHIK was generated in rabbits  and affinity purified. Mouse embryonic fibroblasts grown on coverslips were immunostained with HP1α  and Tif1γ antisera simultaneously. In brief, cells were fixed in 4% paraformaldehyde for 5 min, washed with phosphate-buffered saline, and blocked with 5% serum (PBST) for 30 min. After incubation with the primary antibodies  cells were washed three times with PBST and incubated with secondary antibodies  followed by three washes in PBST. Cells were embedded with Vectashield/DAPI and analyzed using an Axioplan 2 microscope . Digital images were processed using the Volocity 1.0 software . G1E cell differentiation experiments were performed essentially as described .gata1:GFP transgene into 2-d-old embryos reconstitutes erythropoiesis, but not viability, in montg234 homozygous mutants. Movies of live embryos at day 3 posttransplant highlight less than 100 GFP+ RBCs in circulation. Transplanted cells were observed to proliferate, resulting in thousands of donor-derived erythrocytes 7 d later. Movies present GFP-fluorescent images of live zebrafish larvae.Transplantation of wild-type zebrafish marrow cells carrying a Video S1(13.7 MB MOV).Click here for additional data file.Video S2(11.3 MB MOV).Click here for additional data file.Video S3(7.9 MB MOV).Click here for additional data file.Video S4(11.2 MB MOV)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genes and gene products discussed in this paper are fly bonus (AAF19646), human TIF1α (015164), human TIF1β (Q13263), human TIFγ (Q9UPN9), human TIF1γ (Q9UPN9), mon (AY59853), mouse Tif1α (Q64127), mouse Tif1β (AAH58391), and mouse Tif1γ (NP444400).The GenBank (mon/tif1γ and tif1α have been deposited in GenBank under the accession numbers AY598453 and AY598454, respectively.The cDNA sequences of zebrafish


This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels.Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for β-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion.While exploring pharmacological properties of alphaOur results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology. Because the BLA regulates a number of anxiety- or fear-related behaviors [2β compounds possess an appropriate therapeutic index, the pharmacology of these forebrain glycine receptors must be elucidated and extensively compared with the spinal isoform.It has been well established that the amygdala is important in the acquisition and maintenance of fear/anxiety-related behaviors . Strychnsitivity . This fipression . A similrneurons . It is qehaviors , it is p1 expressed in Xenopus ooctyes [1 expressed in HEK 293 cells [1, taurine efficacy is even weaker for GlyRα2 (ca. 5–10% efficacy) when expressed in Xenopus oocytes [2β receptors expressed by BLA neurons possess >50% efficacy for taurine and almost full efficacy for β-alanine [There have been conflicting reports regarding the details of glycine receptor pharmacology when expressed in heterologous systems. For example, taurine acts as a partial agonists (ca. 50% efficacy compared to glycine) for GlyRα ooctyes  whereas 93 cells . Compare oocytes . However2 and α2β receptors since these appear to be the predominate isoforms found in the embryonic and adult forebrain, respectively. Our results provide potentially important insight into the types of mechanisms that may govern brain region-to-brain region variation in glycine receptor pharmacology. Several aspects of this work have appeared in abstract form [This study utilizes whole-cell patch clamp electrophysiology to examine the influence of distinct heterologous expression systems on the β-amino acid pharmacology of glycine receptors composed of distinct subunit combinations. We have focused on the αact form ,12.2, and GluRα2/β in HEK-293 cells and in L-cell fibroblasts. Glycine-gated responses for each receptor isoform were elicited in a dose-dependent manner in both cell types  and α2β (n = 7–8), respectively. GlyR subunits expressed in L-cells displayed a similar pharmacological profile. However, the apparent glycine EC50 of both GlyRα2  and GlyRα2β  appeared lower than apparent affinities for the same subunits when expressed in L-cells. Two-way ANOVA on the Log (EC50) values  and 570 μM (n = 7) for α2 and α2/β receptors, respectively. In L-cells, β-alanine also elicited currents in a dose-dependent manner. And, like glycine, β-alanine appeared to be less potent in these cells compared to HEK cells. EC50 values for α2 and α2/β receptors were 2.0 mM (n = 8) and 2.9 mM (n = 7), respectively. Two-way ANOVA on LogEC50 values from these studies indicate a significant effect of the expression system on β-alanine potency . There was a trend for the presence of the β-subunit to influence potency but this was not significant nor was there any significant interaction between expression system and subunit composition.With the glycine pharmacological profile established, we next examined the pharmacology of the partial agonists, β-alanine and taurine. The efficacy and potency of these β-amino acids were compared to glycine by normalizing the current response at each concentration to a maximal glycine response in that same cell. In HEK cells Fig. , the ave2 and α2β isoforms had efficacies of 80 ± 6% and 55 ± 7% of the maximal glycine response, respectively. A similar trend was noted in L-cells with the α2 and α2β isoform with β-alanine efficacies being 39 ± 8% and 25 ± 5% of the maximal glycine response. Two-way ANOVA analysis of these data indicate that both expression system and subunit composition had a significant influence on β-alanine efficacy . There was no significant interaction between these variables. These data demonstrate that the presence of the β subunit reduced β-alanine efficacy of α2-containing receptors and that this efficacy was substantially smaller L-cells compared to HEK cells.β-alanine efficacy was also examined in these same experiments by normalizing the maximal β-alanine response as a fraction of a maximal glycine response. In HEK cells, the α50 for taurine was 501 μM for GlyRα2 (n = 9) and 2 mM for GlyRα2β (n = 7). Because of its remarkably low efficacy in L-cells (see below), we can only provide estimates of taurine potency in this expression system. Regardless, apparent taurine affinity for both GlyRα2 and GlyRα2β expressed in L cells were ~3 mM for both isoforms . We did not compare L cell data with that obtained from HEK cells due to the uncertainty surrounding the fits. However, there was no significant difference in apparent taurine potency between the α2 and α2β receptors expressed in HEK cells .Similar analysis of taurine pharmacology in HEK and L-cells revealed more dramatic effects of system and subunit on this partial agonist Fig. . In HEK 2 and 32 ± 4% of glycine for the GlyRα2β isoform. Efficacy for these same receptors was reduced to approximately 6 ± 1% and 5 ± 0.7% of glycine when they were expressed in L-cells in these particular studies. The system difference was significant with two-way ANOVA  with no substantial effects of subunit composition or interactions between these variables.Taurine efficacy was obviously quite different between the two expression systems. In HEK cells, taurine efficacy was 48 ± 12% of glycine for GlyRα2 protein using western analysis of total lysate derived from the same number of GFP+ HEK 293 or L cells from transfected cultures , where res Fig. . For thi2 and α2β receptors expressed in HEK cells compared to receptors expressed in L cells  but not subunit, nor was there a significant interaction between variables. Results from both westerns and functional experiments therefore indicate that relative expression levels of glycine receptor were different between HEK and L-cells.Maximal conductance is an independent measure of functional expression and was also larger for both αlls Fig. . Across 2 and α2β glycine receptors in a number of different heterologous systems, as well as for native receptors expressed in rat lateral/basolateral amygdala. In addition to HEK and L-cells, the heterologous systems included mouse 3T3 fibroblasts and MDCK kidney cells. α2β receptors expressed in mouse 3T3 fibroblasts had twice the current density (121 ± 34 pA/pF) of the mouse L-cells (59 ± 19 pA/pF) but had a similar taurine efficacy (13 ± 8% of glycine in 3T3 cells versus 8 ± 1% in L-cells). Similarly, α2β receptors expressed in HEK293 cells had a current density similar to GlyRs expressed in 3T3 fibroblasts (115 ± 11 pA/pF) but had a taurine efficacy compared to glycine of 48 ± 3%. This efficacy was similar to glycine receptors expressed by acutely isolated adult rat basolateral amygdala neurons (46 ± 5% of glycine) although the current density in this native system was only 57 ± 14 pA/pF. Note that the channels expressed by these neurons are composed primarily of α2+β subunits [2β current density (15 ± 5 pA/pF); yet the channels expressed by this system had the highest taurine efficacy of any cell tested (101+6%). For α2 GlyRs, the rank order of glycine receptor density was 3T3 (111 ± 21 pA/pF)> HEK cell (90 ± 11 pA/pF)> L-cell (50 ± 9 pA/pF); while the rank order of taurine efficacy for these same receptors was HEK (74 ± 9%)> 3T3 (23+7%)> L-cells (11 ± 2%). Across all subunit combinations and systems, there was no significant correlation  between taurine efficacy and glycine current density . Indeed, no correlation between expression level and taurine efficacy was evident within any given population of cells whether the receptors were expressed in native or heterologous systems. For example, the correlation coefficients for α2β receptors between taurine efficacy and glycine current density in individual systems were 0.11, 0.13, 0.19, 0.05, and 0.31 for HEK, L-cells, 3T3 cells, MDCK cells, and amygdala neurons, respectively (P >> 0.05). Thus, while there is clearly a difference in expression level between both the systems as well as between individual cells in a given system, this particular characteristic cannot account for the apparent taurine efficacy.To further explore the interaction between expression level and partial agonist efficacy, both current density and taurine efficacy were compared for αy 121 ± 3 pA/pF of50 concentration of glycine. The strychnine KB was estimated for HEK and L-cells expressing either the GlyRα2 or GlyRα2+β subunits using the Cheng-Prusoff relationship (see Methods). This relationship takes into account the divergent Hill-slope and potencies for glycine found in these two expression systems. Receptors composed of the GlyRα2 subunit  or L-cells  in HEK cells and 38 ± 8 nM (n = 10) in L-cells. Two-way ANOVA did not reveal any significant effect of either system or subunit composition. Since strychnine is a competitive antagonist and site-directed mutation studies suggests that strychnine and glycine interact with overlapping regions of the receptor [There are a variety of possible mechanisms to account for the disparities in partial agonist pharmacology between two expression systems. One way to address this is to examine competitive antagonist binding properties in HEK and L-cells. We therefore examined the potency of the glycine receptor competitive antagonist strychnine in both systems. Following a 30 second pretreatment with the antagonist , we co-anit Fig.  had veryreceptor -16, our 50 concentration of glycine (300 μM) did not significantly affect glycine-gated currents themselves. Glycine currents were 17.3 ± 2.3 pA/pF while glycine+colchicine currents were 16.7 ± 2.2 pA/pF .A third possible mechanism for reduced efficacy in L-cells compared to HEK cells or neurons could be related to intracellular factors that influence channel gating . This hy2β subunits following 30 min incubation with 100 μM colchicine at 37°C, enough time to allow irreversible tubulin disruption [2β-expressing HEK cells  and β-alanine  were significantly decreased by colchicine but not γ-lumicolchicine treatment. Taurine efficacy was 33 ± 6% of glycine in controls, 13 ± 3% following colchicine, and 28 ± 3% following γ-lumicolchicine. Similarly, β-alanine efficacy was 70 ± 7% of glycine in controls, 49 ± 6% following colchicine, and 72 ± 7% following γ-lumicolchicine. Similar treatment of α2-expressing HEK cells with colchicine  but not in GlyRα2-expressing L-cells  and 'forebrain' (α2β heteromeric) isoforms in two distinct expression systems to understand the influence of endogenous and exogenous factors on receptor partial agonist pharmacology. Although the pharmacology of the 'embryonic' GlyRα2 isoform and the 'adult spinal' isoform (GlyRα1β) have been explored more frequently in the literature, the pharmacology of GlyRα2β receptors has remained largely unexplored. Despite this, there is strong evidence that the adult 'forebrain' isoforms, specifically in the rat basolateral amygdala, is indeed α2β [2β subunits compared to their homomeric α2 counter parts.We have expressed several the 'embryonic'  replaced the β subunit when examining α2 homomeric channels.HEK 293 , mouse L-cells , NIH/3T3 fibroblasts (ATCC), and MDCK cells  were grown in Dulbecco's modified Eagle's medium  with 10% fetal bovine serum  and 1X penicillin/streptomycin (Life Technologies) on Thermanox cover slips in 35 mm culture dishes. Cells were transfected during log-phase growth (30–50% confluent) using the Superfect reagent , according to manufacturers instructions. Rat glycine receptor αeviously . Briefly2 1, Mg-ATP 4, pH 7.2 with methane sulfonic acid; adjusted to 290–295 mmol kg-1 with sucrose. Whole cell capacitance and series resistance was manually compensated after opening the cell. Cells were continuously bath perfused with a HEPES-buffered saline (in mM): NaCl 150, Glucose 10, HEPES 10, KCl 2.5, CaCl2 2.5 MgCl2 1.0, pH 7.4, 305–320 mmol kg-1 Data will be analyzed off-line using pClamp software (Axon). Numerical analysis was performed using commercially available software. Independent student's t-test and two-way ANOVA were used for comparisons where appropriate; and statistical significance was based on p < 0.05. Concentration-response curves were generated from fits of data to a standard logistic equation as previously described . To derive KB from functional IC50 and EC50 data, the Cheng-Prusoff equation was used:Whole cell recordings were performed at room temperature using standard patch-clamp techniques and the axopatch-1D amplifier  in the voltage clamp mode. Gigaohm seals were formed using patch pipettes made from borosilicate glass . For most experiments, the internal solution contained (in mM): CsCl 100, EGTA 11, HEPES 10, CaClS = hillslope of agonist curve, A = concentration of agonist, EC50 = half-maximal agonist concentration, and IC50 = half-maximal antagonist concentration.where Stocks of glycine, taurine, β-alanine, strychnine (Tocris) and colchicine (SIGMA) were prepared fresh each day. Agonists and antagonists were applied for 4–10 sec from an array of eight HPLC-grade capillary tubes  placed within 100 μm of the cell of interest.Cells were cultured, transfected as stated above except in 10 cm Petri dishes, and harvested by scrapping with 500 μL of lysis buffer . Proteins were quantified using the Bradford assay and loaded onto 8–10% SDS-polyacrylamide gels, separated, and transferred to a nitrocellulose membrane . The membrane was blocked overnight TBS  containing 0.2% Tween-20 and 10% low-fat dry milk. Blots were then incubated in TBS/0.1% Tween-20 containing primary GlyR antibody  and monoclonal anti-gephyrin antibody  for two hours at room temperature. After several washes, the HRP-coupled rabbit anti-mouse secondary antibody (SIGMA) was added for one hour (1:2000). The detection was performed by the ECL method (Amersham).GlyR – glycine receptor; BLA – lateral/basolateral amygdala; HEK – human embryonic kidney cells; MDCK – Manin-Darby canine kidney cellsJF carried out the electrophysiological recordings and participated in the western analysis. BM conceived of the study, participated in its design and coordination, performed the western analysis and some electrophysiology experiments, and drafted the manuscript. All authors read and approved the final manuscript.