A Retrospective by Adolfo J. de Bold Journal of the American Society of Nephrology [With the permission of A. J. de Bold] The discovery of an endocrine link between the heart and the kidneys has its basis in the electron microscopic finding that the striated muscle cells of the cardiac atria in mammals are differentiated both as contractile and as endocrine cells. The demonstration that atria produce polypeptide hormones was established with the discovery of atrial natriuretic factor (ANF). ANF is the founder member of the ANF family of natriuretic peptides that have very important functions in the modulation of volume regulation and cardiovascular growth. The unfolding of this discovery, as many others, has a great deal of human content that often is lost in our technical writings. I hope that students and investigators who are just starting out will find inspiration (and consolation) in the informal account of the ANF discovery that follows. When I arrived to the pathology department at Queen's University [Kingston] in 1968, fresh from obtaining a degree in clinical biochemistry from the Faculty of Chemical Sciences in Cordoba, Argentina, my supervisor, Sergio Bencosme, was interested in the functional morphology of the endocrine pancreas. As an aside, Bencosme had taken up the question of secretory-like differentiations found in atrial cardiocytes, a fact known since the early days of electron microscopy and manifesting itself most notably by the presence of storage granules known as "specific atrial granules" whose function was a mystery. He and many other notables, including George Palade then at the Rockefeller Institute in New York, could not advance past their morphologic description. Others considered the atrial granules as an evolutionary remnant. I found myself unable to ignore a secretory phenotype and made it a personal challenge to demonstrate that a combination of morphologic and biochemical techniques would unravel the functional nature of the atrial granules. Perhaps I was influenced by the great endocrine work of Argentinean Nobel Prize laureate B. Houssay, who is an icon of academic excellence for anyone born in Argentina. And so the ANF saga began. It would take 12 yr of investigations (with only 1 month of holidays) before the nature and function of the dual secretory-contractile nature of atrial cardiocytes would become apparent (for a review, see reference [1] ). I began my studies on the possible secretory function of the heart by trying to isolate the atrial granules armed with the papers produced by Christian De Duve on isolation of subcellular organelles and the paper by Blascho on isolation of adrenal chromaffin granules. There were literature data that hinted that the atrial granules were a storage site for catecholamines, but a careful read of the literature was not very convincing in that sense. At any rate, this was a hypothesis to test and this turned into my project for my master of science project. The isolation of the granules was particularly difficult because they were immersed in the great tangle of myofibrils and connective tissue that represents a homogenate of the heart muscle. Therefore, it took me 2 yr and quite a few 20- to 60-rat ultracentrifugation runs to obtain the purified granules. Bigger animals (cow hearts were suggested many times) were of no use because there is an inverse relationship between the number of atrial granules found in atrial cardiocytes and the size of the animal. Because I had no biochemical marker for the granules, the most tedious job that I found was to look at every fraction by electron microscopy to see where the granules went with the many variations to the isolation technique [2] . For this purpose I developed an electron microscopy embedding technique to deal with subcellular fractions. After many trials, I was able to isolate and purify the granules and proved by biochemical means that the granules did not contain catecholamines [3] . This was success in one sense, but it also meant that I had no hypothesis left to test. I set out to develop techniques to visualize specifically the atrial granules at the light microscopic level. I reasoned that with such a technique, one could correlate the distribution of the granules with histochemical reaction products. It helped me enormously that, by intervention of Divine Providence I am sure, I had managed twice during my undergraduate years to end up working as a research assistant in pathology departments, where I learned many histologic techniques. I developed the first method to stain specifically the granules at the microscopic level using lead-hematoxylin following a paper that my wife had found to stain cells in the pituitary gland [4]. The stain aldehyde-fuchsin also provided a visualization of the granules. With these techniques at hand, I carried out a whole battery of histochemical investigations [5]. A number of cytochemical properties of the atrial granules thus were uncovered. These investigations would later help me to isolate and purify ANF. For example, the poor stainability of the atrial granules following Bouin's fixative (a fixative that contains acetic acid) suggested that the granules' content was soluble in acetic acid. Indeed, ANF and brain natriuretic peptide are highly soluble in acetic acid, which is the basis for extractants of these hormones. Altogether, these cytochemical studies plus the ones that I carried out later as an independent investigator provided evidence that the atrial granules stored a random-coiled, basic polypeptide that contained cystine and tryptophan. Autoradiographic studies with radiolabeled leucine showed that the content of the granules had a high turnover in a manner similar to secretory cells [6]. All of these properties were confirmed later by biochemical means following the isolation of ANF. By this time (1973), I had finished my doctoral thesis, my first of our five children had been born, and we had purchased our first home. I was then offered a position to continue at Queen's, moving to the Pathology Laboratory at Hotel Dieu Hospital, a teaching hospital associated with Queen's University, as an assistant professor of pathology. I was to help develop research at this hospital, and it was a leap of faith of the chairman of pathology, Nathan Kaufman, to put me there and for which I am very grateful. Years later, after the discovery of ANF, Nate reminded me that during my thesis defense, I had guessed that the atrial granules, because of their location, might be involved in sensing changes in volume load. I had forgotten that. A service-oriented hospital, Hotel Dieu was not the most propitious place for a young scientist. I was given an office, half a lab bench, an old incubator, and a microscope to start. Mine was a windowless office in the basement, across from the autopsy room. The smell of formalin was a constant companion. Looking back, this isolation helped me in continuing with the goal of establishing the endocrine function of the heart. My first grant application to the Medical Research Council as an independent researcher was on the status of the cardiac adrenergic innervation in heart failure. This is the reason that I have a publication on a new model for inducing heart failure in the guinea pig. This theme was really a safety net in case the atrial granule business did not work out. As it turned out, the reverse occurred. I secured funding from the Heart and Stroke Foundation to continue my graduate studies on atrial granules, but I knew that their patience was wearing thin on this theme. I also collaborated with Jack Kraicer of the physiology department at Queen's on the morphology of the pars intermedia of the pituitary gland. My wife, who had started working with me, and I were able to define a system of canaliculi in this avascular gland using extracellular space markers. In the process, we discovered a new cell type for which my wife developed a silver impregnation technique to demonstrate it at the light microscopic level [7]. Although the nature of the granule's content seemed reasonably well defined by the histochemical studies, there was still no hint as to their function. However, we now had something that was not available previously. Namely, a stain that could demonstrate the granules at the light microscopic level; therefore, we could develop a quantitation procedure to assess changes in the number of granules after different experimental procedures using the light microscope. The difference between a morphometric procedure at the light microscopic level and procedures at the electron microscopic level is that the sample size is made much larger at the light microscopic level. This was particularly important for quantitation of atrial granules because of their irregular distribution in the atria and even within the same cell. We developed a morphometric procedure using the light microscopic staining developed during the histochemical studies using embedding in plastic to obtain uniformly thin sections of atrial tissue. Such a procedure then was tested statistically [8] and was used later to test claims that previous researchers had made regarding the ability of certain experimental maneuvers to change the number of granules. There were many such claims and counter claims, and I tested most. I found unequivocal, statistically significant changes in the number of granules after some procedures that were known to alter water and electrolyte balance as previously suggested in electron microscopic studies by Bencosme and Hatt (reviewed in reference [9]). The difference afforded by the light microscopic quantitation of granules was that one could be confident that the changes were not the result of biased sampling, and therefore one really could commit one's time to further the study without the feeling of being wasteful. The hypothesis thus developed was that the atria produced and stored a polypeptide that helped regulate water and electrolyte balance given the nature of the contents revealed by histochemistry and the changes in the number of granules revealed by the morphometric technique after procedures that were known to alter water and electrolyte balance. I thought that the easiest way for a cardiac hormone to modify water and electrolyte balance was to target the prominent role of the kidneys in maintaining water and electrolyte homeostasis. Besides, the atria were in an ideal spot to sense changes in venous return. Looking for a bioassay for diuretic substances, I found that Harald Sonnenberg of the Department of Physiology at the University of Toronto, whom I did not know, was searching for a natriuretic hormone and had a rat bioassay for that purpose. I phoned him and related to him my quest and hypothesis. Because the existence of atrial granules was not widely known, even by morphologists, it was specially generous of Harald to accept my invitation "to take a shot in the dark." He invited me to give a seminar in Toronto, and we agreed that I would send him atrial extracts. The first extracts were, in fact, atrial granule extracts that contained a high concentration of potassium chloride because of the composition of the solutions used for isolation. This promptly killed the bioassay rats upon injection. I then more or less supplicated Harald to be patient and please to try just crude extracts of atria, and of ventricles as a control, prepared in simple phosphate-buffered saline. Some weeks went by, and then, to my unbelieving ears, Harald phoned me to say that the injection of atrial extracts produced a diuresis and natriuresis that was immediate and incredibly strong, just like furosemide. Always a worrier, I started to wonder about what contamination would produce such effects. We repeated the experiments many times in my lab, and the results were equally impressive. Also, proteinase destroyed the activity, which went right along with the hypothesis that the atrial granules contained a polypeptide hormone. The potential importance of the finding prompted us to send our findings to the prestigious Journal of Clinical Investigation. It was tersely rejected in a letter dated May 28, 1980, because the finding "was not thought to be suitable for publication." Because I had disclosed the findings previously at a meeting of the Canadian Society for Clinical Investigation, we decided to publish the findings as quickly as possible. For this reason, it was sent to Life Sciences, where it was quickly accepted and published in 1981 [10]. By 1983, the first publications on ANF from other centers started to appear. Not a single laboratory failed to confirm our findings, given that the natriuretic and diuretic activities of atrial extracts were so powerful that nothing short of a dead bioassay rat could stop such action. The article in Life Sciences [1981] spurred a flurry of activity and went on to become a Citation Classic as qualified by the Institute of Scientific Information. Needless to say, the researchers in the hypertension field were ready to exploit the finding of a hormone that was diuretic, natriuretic, and hypotensive. It is of interest to note the different reactions by different groups of investigators. Some invited me to present my work and recognized the discovery in one way or another. Others embarked on ANF in furious research and in public relations campaigns, some including televised speeches, to convince the world that they had discovered ANF. It was never clear to me how they planned to claim a discovery for which we had an indisputable 3-yr precedence in publishing. I guess that a discovery that came from a basement of an obscure hospital was deemed easy prey. At any rate, it seems that all discoveries follow the same libretto. The Japanese authors, although they also invented a new name (ANP) and thus disregarded an international nomenclature agreement reached in New York and still existing, truly did contribute to the natriuretic peptide field by demonstrating the occurrence of brain natriuretic peptide and C-type natriuretic peptide based on the ANF discovery. Our laboratory also was the first to isolate, purify, and sequence ANF [11] [12] [13]. The way that this was accomplished was not less heroic than the 12 yr of work that preceded the ANF discovery. It was very opportune for me to find in the United States a company that provided us with rat atria. In total, approximately 200,000 rat atria were used. It was also fortunate that the techniques for isolation of peptides by HPLC were coming into use. The only problem was that I did not have an HPLC. The clinical laboratory in our hospital, however, had just purchased one to measure theophylline in serum. Luckily, I was put in charge of that technique, so it was not very noticeable that I came during the night to reconfigure the machine and fitted it with a chromatographic column to purify peptides. Three people essentially did the isolation and purification of ANF in my laboratory: my wife would extract the atria, I would purify the extracts, and a technician would test the different fractions obtained during purification in the bioassay rat. No other resource or person was involved in this effort. Once the peptide was purified to chemical homogeneity, my next problem was to sequence it. The only person at Queen's involved in amino acid analysis and protein sequencing was Geoff Flynn, to whom I offered collaboration. We had various false starts because of antiquated equipment, both in the amino acid analysis and in the sequence results. The problems were resolved when we obtained funding from the government of Ontario to purchase a gas phase sequencer; thus, we were the first laboratory to produce a sequence in 1983 [13]. The Japanese workers produced the human sequence the following year. Students often ask for advice to succeed in research, and my standard answer is, "Have a dream, don't think small, work hard, and believe in yourself." I finish this in my mind with, "...and pray that you are right." References1. de Bold AJ, Bruneau BG: Natriuretic peptides. In: Handbook of Physiology, Section 7: The Endocrine System, Volume III: Endocrine Regulation of Water and Electrolyte Balance, edited by Fray JCS, Goodman MH, New York, American Physiological Society by Oxford University Press, 2000, pp 377-409
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Periander sent a messenger to Thrasybulus to ask for advice on ruling Corinth. Thrasybulus did not answer, but took the messenger out for a walk in the corn field. As they strolled along, Thrasybulus idly swatted the corn with his stick, so cutting back the stems that stuck out above the rest. The messenger returned and told Periander what had happened. Periander deduced that Thrasybulus's advice was to kill the most outstanding citizens. Any student of modern tyranny knows that Thrasybulus's advice is followed to this day. The most outstanding citizens are likely to be the prime challengers of the tyrant's power. For more of the significance of this is the present context (Click Here) |
Scullduggery - L'affaire Olivieri (Click Here)
| Election 2000 - Just Ice Those inclined to believe in the probity of high councils, such as those that disburse a nation’s health research funds, were driven from complacency in the fall of 2000 when one of the highest councils, the Supreme Court of the United States of America, handed the Presidency to the republican son of a previous republican President. The republicans had discarded their most competent candidate, Senator John McCain, who strongly advocated reform of campaign financing (see above), and selected George Bush, the Governor of Texas. The democrats selected the Vice-President, Albert Gore. In a time of "unprecedented prosperity" things looked good for Gore, whose 8 year understudy had amply prepared him to assume the highest office in the land. Yet, his style was somewhat "stiff," and his mentor, Clinton, had erred in his private life. By pressing for Clinton’s impeachment on this matter, the republicans had attempting to cripple the Presidency (and hence the important role it had to play in a dangerous world) in support of their own political interests. Bush played the "integrity" card for all it was worth, and the election became "too close to call." Everything came to depend on the vote in Florida, whose 25 electoral college votes would give victory. In the initial count, Bush had a slight lead. However, malfunctioning voting machines in certain democratic counties had rejected large numbers of votes ("undercounts"). The voters in those counties had thus been denied "the equal protection of the law" that the US Constitution’s 14th Amendment required. Gore appealed to the Supreme Court of Florida, which has final authority in state matters, requesting a recount in these counties, and called upon Bush to join him and press for a rapid hand recount of all rejected votes in Florida, so that "the will of the people" would become apparent, and the future President would be legitimized. Bush decline to do this and the matter went to the Supreme Court of the USA. Meanwhile, the State Supreme Court, under pressure from the Federal Supreme Court not to "make new law" gave the go-ahead for the recount, which under existing Florida Law required that the counters manually examine the voting cards to discern "the clear intent" of the voter. While this criterion might differ from counter to counter, an individual counter would use the same criterion whether judging an undercount for Bush or for Gore. So the counting was essentially fair, and could be made fairer by group evaluation of every undercounted ballot. The Florida Law did not elaborate, in advance, what would constitute "the clear intent," thus, wisely leaving the door open to the unexpected (such as a voter leaving the card untouched, except for writing, say, "I vote for George Bush"). In any case, changing that law was a legislative, not a judicial, task. Furthermore, all parties knew the rules when the "game" began and, however imperfect the rules, it was not appropriate to change them when the game was in progress. Amazingly, on Saturday 9th December, the Federal Supreme Court, issued a ruling that the counting be stopped, on the grounds that it would do "irreparable harm" to Bush in that there would be a "cloud" over "the legitimacy of the election". As pointed out by Ronald Dworkin (NY Review of Books Vol 48, no. 1. p. 53-55): This
Even more amazing was the further Federal Supreme Court adjudication that the Florida Supreme Court’s "clear intention of the voter" standard for manual recounts violated the equal protection clause. Given this, it was still possible for the Florida Supreme Court (i) to define a new standard (an essentially legislative act, which the Federal Supreme Count now countenanced) and (ii) to have the manual recount completed by 18th December, when the Electoral College voted. However, the Federal Supreme Court considered that the Florida legislature would want to take advantage of the Federal "safe harbor" law that guarantees immunity against the unlikely event of a Congressional challenge to the Florida result, if the counting is completed by 12th December. Dworkin pointed out:
Knowing that there were time limits, the republicans took every opportunity to delay. The Federal Supreme Court gave its final judgement only 2 hours before the 12th December "safe harbor" deadline expired, stating that the recount would have to be completed by that date. This was, or course, impossible, so that the Presidency was handed to Bush. A dissenting Supreme Court judge observed that the decision "can only lend confidence to the most cynical appraisal of judges throughout the land." Another dissenting judge considered this "self-inflicted wound … may harm not just the court, but the nation". Naked power had won over principle. "Justice" was revealed as just ice, which melts when the political temperature rises. When Gore conceded, many sighed that at least there had been a peaceful transition. But what is a "peaceful transition"? Did they really imagine that there would be a military coup in the USA?
A political transition that leads Ronald Dworkin, Professor of Law at New York University and of Jurisprudence at University College, London, to write as quoted above, may turn out to be far from peaceful. This lack of peace may not be confined to North America. A clear signal has been sent to those who have power, but hesitate to use it. Winning is more important than justice. The ends justify the means. |
Donald Forsdyke. 7th. Jan. 2001.
Time-line adapted from the NY Times
| Tuesday 7th November. Election Day Tuesday 21st November. Florida Supreme Court lets recounts proceed in four counties. Wednesday 22nd November. Bush appeals to US Supreme Court. Friday 24th November. US Supreme Court accepts appeal. Friday 1st December. Case is argued to US Supreme Court. Monday 4th December. US Supreme Court unanimously decides to vacate the Florida Supreme Court's decision, asking for a better explanation of the ruling. Friday 8th December. Florida Supreme Court overturns a lower court decision and order a statewide recount of ballots for which machines had registered no choice for president. Saturday 9th December. Justices accept Bush appeal and, by 5-4 vote, also grant the Bush request to stop the recount in the meantime. Sunday 10th December. Both sides file briefs. Monday 11th December. Cases are argued before the US Supreme Court. Tuesday 12th December. The US Supreme Court rules, 5-4, that the recount as structured by the Florida Supreme Court is unconstitutional and there is no time to fix it. Wednesday 13th December. Gore concedes. |
Legitimacy of the President?
In the final hours of his administration the out-going President took an important step to improve the legitimacy of Bush. The NY Times declared on 24th Jan:
Casualties: First Justice, then Truth?
F rom all this it is hard not to suspect that, just as the real experts who might have given advice on bioterrorism were sent "off fishin" because of the way our health research peer-review system works, so (?)President Al Gore (and his probable Secretary of State, Richard Holbrooke) were sent "off fishin" because of the way the US electorial system works.At times which demand the very best of the very best, we should know if our world is in the hands of Governor George Bush or President George Bush? He states that, while being cautious, US citizens should get on with their lives and show the terrorists that they cannot succeed. However, "business as usual" requires that the press be unshacked. Donald Forsdyke, 14th October 2001 On 12th November, the press were unshackled. Yes, if all the Florida votes had been counted Gore would have won by from 42 to 171 votes. However, the headlines did not reveal this and one had to dig deep to find the truth. Gore's requests for limited recounts had been rejected, so it showed good judgment that he had requested, but not pressed for, a state-wide recount. If the limited recount proposal were rejected than it is certain that the state-wide recount would also have been rejected. It turned out that, if only the limited recounts had been performed, then Bush would have maintained a slight lead. But that detracted from the real issue. Governor Bush was now in charge, for better or for worse. The most valid question was, who got the most votes in Florida? The result, albeit through a "photo-finish", was President Gore. There was much mumbling about the result being statistically insignificant. Well, so is the result of a photo-finish in the 100 metres dash. Yet, to the winner we accord the gold. Political contests are won by those who get a nose ahead. Statistics are irrelevant. Donald Forsdyke 12th November 2001 |
Demographic Shift in the Research Community
From all the above, you should be getting an increasingly clear idea of the role of politics, and particularly drug politics, in the decline in the health research system.
The result has been a demographic shift, over several decades, in the academic composition of our universities and research institutes. While there should be a place for all types of researchers and all styles of research in these institutions, now one type and style prevails.
Would Charles Darwin have survived in the modern research environment? Well, he was independently wealthy and did not have that problem. But wouldn't it be nice to have Darwin around today to advise on AIDS! The days of the wealthy "gentleman scientist" are long past. Most researchers are obliged to enter the funding marketplace, which then coerces the direction their research takes.
This situation will prevail as long as:
The pervasive influence of "the dollar" is everywhere. Just as it is ability at campaign fund-raising, rather than ability to govern that decides our political leaders (see above), so Boards of Trustees elect University Principals or Presidents based on their fund-raising potential, rather than on their potential for wise governance. When a University Department or Research Institute considers the Curriculum Vitae of a possible recruit the major question asked is "Is he/she fundable?" not "How able and innovative is this person?" The likes of Irvine Page, Szent Gyorgyi and Erwin Chargaff have disappeared from view. When your son or daughter expresses disappointment about his/her university experience, think not that the fault lies with the complainant. That he/she is inclined to drop-out, may reflect the fact that the chances of finding teachers of caliber are now greatly reduced (although thankfully not entirely eliminated). For how this is playing out tragically in the case of AIDS in South Africa Click Here.However, the implications of this go far beyond the management of natural diseases. Currently, the greatest threat to humankind seems to be overt or terrorist warfare conducted not with nuclear weapons, but with biological weapons. A nation which uses the peer-review process, as it currently (200 2) operates, to select those who give it advice on biomedical matters, may not fare well in confrontation with a nation which has adapted the peer-review process to identify those (e.g. Irvine Page, Szent-Gyorgyi, Erwin Chargaff) who can see beyond their noses. For more: Click HereWhen the author of these web-pages presented some of the above views to the Canadian Standing Parliamentary Committee on Industry, Science and Technology (29th Nov. 2001;(Click Here)), one of the nation's elected representatives suggested he was engaging in hyperbole! How many more scandals at The Hospital for Sick Children, how many more Fabricant shootings, must there be, before it is appreciated that the peer-review emperor might be less than fully clad? The rigidity of the system parallels only that of the financial empires which rule our private enterprise system. In an article entitled "The Betrayal of Capitalism" Ambassador Felix Rohatyn pointed to the failure of peer-review when yet another scandal (Enron) rocked the USA (New York Review of Books 49, #3; Feb. 2002):
One by one, the idols fall and public disillusion increases.
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