Can we see living structure in a cell?

Colloid chemistry (κολλα: glue, or gelatin) was introduced in 1861 after the discovery of protoplasm, which exhibits gelatin-like properties. Some 80 years later, colloid chemistry (and with it, the concept of protoplasm) was largely abandoned. The membrane (pump) theory, according to which cell water and cell solute like K+ are free as in a dilute KC1 solution, became dominant. Later studies revealed that rejecting the protoplasmic approach to cell physiology was not justified. Evidence against the membrane (pump) theory, on the other hand, has stood the test of time. In a new theory of the living cell called the association-induction (AI) hypothesis, the three major components of the living cell (water, proteins and K+) are closely associated; together they exist in a high- (negative)-energy-low entropy state called the living state. The bulk of cell water is adsorbed as polarized multilayers on some fully extended protein chains, and K+ is adsorbed singly on ß- and γ-carboxyl groups carried on aspartic and glutamic residues of cell proteins. Extensive evidence in support of the AI hypothesis is reviewed. From an extension of the basic concepts of the AI hypothesis and the new knowledge on primary structure of the proteins, one begins to understand at long last what distinguishes gelatin from other proteins; in this new light, new definitions of protoplasm and of colloid chemistry have been introduced. With the return of the concept of protoplasm, living structure takes on renewed significance, linking cell anatomy to cell physiology. Finally, evidence is presented showing that electron microscopists have come close to seeing cell structure in its living state.

A 2004 unanswered letter to the Economist magazine requesting a retraction (and apology).

This is a copy of (the bulk of) a letter I mailed on May 13, 2004 to Sir Robert P. Wilson, President, and three editors of the magazine, the Economist. With the letter, I also sent each recipient a copy of my latest book, "Life at the Cell and Below-Cell Level" as a gesture of good will.

Truth in basic biomedical science will set future mankind free.

It is self-evident that continued wellbeing and prosperity of our species in time to come depends upon a steady supply of major scientific and technologic innovations. However, major scientific and technical innovations are rare. As a rule, they grow only in the exceptionally fertile minds of men and women, who have fully mastered the underlying basic sciences. To waken their interest in science at an early critical age and to nurture and enhance that interest afterward, good textbooks at all level of education that accurately portray the relevant up-to-date knowledge are vital. As of now, the field of science that offers by far the greatest promise for the future of humanity is the science of life at the most basic cell and below-cell level. Unfortunately, it is precisely this crucial part of the (standardized) biological textbooks for all high schools and colleges in the US and abroad that have become, so to speak, fossilized. As a result, generation after generation of (educated) young men and women have been and are still being force-fed as established scientific truth an obsolete membrane (pump) theory, which has been categorically disproved half a century ago (see Endnote 1.) To reveal this Trojan horse of a theory for what it really is demands the concerted efforts of many courageous individuals especially young biology teachers who take themselves and their career seriously. But even the most courageous and the most resourceful won't find the task easy. To begin with, they would find it hard to access the critical scientific knowledge, with which to convert the skeptic and to rally the friendly. For the wealth of mutually supportive evidence against the membrane (pump) theory are often hidden in inaccessible publications and/or in languages other than English. To overcome this seemingly trivial but in fact formidable obstacle and to reveal the beauty and coherence of the existing but untaught truth, I put together in this small package a collection of the major clenching theoretical and experimental findings. These findings will remove the last trace of uncertainty about the total disproof of the membrane theory. In addition, I have also included an introduction of the association-induction hypothesis, which is the one and only unifying theory of the living cell that has survived and unwaveringly grown more comprehensive and powerful after more than half of a century of worldwide testing.

A preliminary report on the survival of fully-hydrated living (cancer) cells to liquid helium exposure.

Fully-hydrated Ehrlich carcinoma ascites cells under the protective action of DMSO fully survived exposure to near-absolute zero temperature provided by liquid helium.

A historically significant study that at once disproves the membrane (pump) theory and confirms that nano-protoplasm is the ultimate physical basis of life--yet so simple and low-cost that it could easily be repeated in many high school biology classrooms worldwide.

In 1889 Abderhalden reported his discovery that there is no (or as shown later, little) sodium ion (Na+) in human red blood cells even though these cells live in a medium rich in Na+. History shows that all major theories of the living cell are built around this basic phenomenon seen in all the living cells that have been carefully examined. One of these theories has been steadily evolving but is yet-to-be widely known. Named the association-induction hypothesis (AIH), it has been presented thus far in four books dated 1962, 1984, 1992 and 2001 respectively. In this theory, the low Na+ in living cells originates from (i) an above-normal molecule-to-molecule interaction among the bulk-phase cell water molecules, in consequence of (ii) their (self-propagating) polarization-orientation by the backbone NHCO groups of (fully-extended) cell protein(s), when (iii) the protein(s) involved is under the control of the electron-withdrawing cardinal adsorbent (EWC), ATP. A mature human red blood cell (rbc) has no nucleus, nor other organelle. 64% of the rbc is water; 35% belongs to a single protein, hemoglobin (Hb). This twofold simplicity allows the concoction of an ultra-simple model (USM) of the red blood cell's cytoplasmic protoplasm, which comprises almost entirely of hemoglobin, water, K+ and ATP. Only in the USM, the ATP has been replaced by an artificial but theoretically authentic EWC, H+ (given as HCl). To test the theory with the aid of the USM, we filled dialysis sacs with a 40% solution of pure (ferri-) hemoglobin followed by incubating the sacs till equilibrium in solutions containing different amounts of HCI (including zero) but a constant (low) concentration of NaCl. We then determined the equilibrium ratio of the Na+ concentration inside the sac over that in the solution outside and refer to this ratio as qNaCl. When no H+ was added, the qNaCl stayed at unity as predicted by the theory. More important (and also predicted by the theory,) when the right amount of H+ had been added, qNaCl fell to the 0.1- 0.3 range found in living red blood (and other) cells. These and other findings presented confirm the AIH's theory of life at the most basic level: in the resting living state, microscopic, or nano-protoplasm, is the ultimate physical basis of life. (See Post Script on page 111.)

Nano-protoplasm: the ultimate unit of life.

Among the most promising scientific achievements of the 19th century was the recognition that the laws governing the dead world also govern the world of the living and that life has a physical basis called protoplasm. Regrettably, the definition of protoplasm provided then was (inescapably) incorrect, offering a (legitimate) reason for rejecting the concept of protoplasm by an overwhelming majority of later investigators, teachers and other opinion-makers. Without a recognized physical basis, Life itself also faded into the limbo of the unexplainable. However, eventually the needed relevant parts of physics and chemistry to give a more cogent definition of protoplasm became available. That then made possible the construction in the early 1960's of a unifying theory of the living cell, named the association-induction (AI) hypothesis. Historically speaking, the AI Hypothesis is the heir to the general concept of protoplasm as the physical basis of life-incorrect as the initial definition of protoplasm was notwithstanding. In the AI Hypothesis (AIH) the true or ultimate physical basis of life is not what the advocates of the protoplasm once considered as the physical basis of life. What they saw and construed as the physical basis of life is a particular kind of macroscopic protoplasm. In the AI Hypothesis, the basic unit (or physical basis) of life is microscopic protoplasm or nano-protoplasm, of which all macroscopic protoplasm is made. The AI Hypothesis also had no difficulty offering a new definition to what life is in terms of fundamental physical-chemical laws. Nano-protoplasm is defined by what it is and what it does. In greater detail, it is defined (i) by its chemical composition given in Equation 1 on p. 124; (ii) by the mutual spatial and energetic relationships among the components as illustrated diagrammatically in Figure 5 on p. 125; and (iii) by the ability of these components to exist as coherent assemblies in either one of two alternative states, the resting and active living (or dead) state as according to Equation 5 on p. 142. The review then describes the AIH-based electronic and molecular mechanisms for the coherent assemblage of the components, for the maintenance of the living states and for the auto-cooperative transitions between the resting and active (or dead) living state. Having completed the theoretical section, the review goes on to describe the experimental testing of the theory carried out in the past forty-some years (and even in time before that by authors who knew nothing of the theory.) These experimental studies fall into two broad categories. In the first category, are the experiments performed on ultra-simple models of nano-protoplasm made up from pure chemicals as prescribed in Equation 1 on p. 124. The results show that they indeed behave qualitatively like that illustrated in Figure 5 and quantitatively follow the dictates of Equation 5. In the second category of experimental testing, parallel studies were carried out on nano-protoplasm as part of living cells--in carrying out each one of the four classical functions of cell physiology: (1) solute and water distribution; (2) solute and water permeability; (3) cellular resting and action potentials; (4) cellular swelling and shrinkage. The results show that the nano-protoplasm in situ too qualitatively behave like that shown in Figure 5 and quantitatively follow the dictates of Equation 5. The review ends on a discussion section, examining how cogent do the experimental data accumulated thus far support to the AI version of the concept of nano-protoplasm as the most basic unit of life.

History of the membrane (pump) theory of the living cell from its beginning in mid-19th century to its disproof 45 years ago--though still taught worldwide today as established truth.

The concept that the basic unit of all life, the cell, is a membrane-enclosed soup of (free) water, (free) K+ (and native) proteins is called the membrane theory. A careful examination of past records shows that this theory has no author in the true sense of the word. Rather, it grew mostly out of some mistaken ideas made by Theodor Schwann in his Cell Theory. (This is not to deny that there is a membrane theory with an authentic author but this authored membrane theory came later and is much more narrowly focussed and accordingly can at best be regarded as an offshoot of the broader and older membrane theory without an author.) However, there is no ambiguity on the demise of the membrane theory, which occurred more than 60 years ago, when a flood of converging evidence showed that the asymmetrical distribution of K+ and Na+ observed in virtually all living cells is not the result of the presence of a membrane barrier that permits some solutes like water and K+ to move in and out of the cell, while barring--absolutely and permanently--the passage of other solutes like Na+. To keep the membrane theory afloat, submicroscopic pumps were installed across the cell membrane to maintain, for example, the level of Na+ in the cell low and the level of K+ high by the ceaseless pumping activities at the expense of metabolic energy. Forty-five year ago this version of the membrane theory was also experimentally disproved. In spite of all these overwhelming evidence against the membrane-pump theory, it still is being taught as verified truth in all high-school and biology textbooks known to us today. Meanwhile, almost unnoticed, a new unifying theory of the living cell, called the association-induction hypothesis came into being some 40 years ago. Also little noticed was the fact that it has received extensive confirmation worldwide and has shown an ability to provide self-consistent interpretations of most if not all known experimental observations that are contradicting the membrane-pump theory as well as other observations that seem to support the membrane pump theory.

An unanswered 2003 letter appealing on behalf of all mankind to Nobel Laureate Roderick McKinnon to use his newfound fame and visibility to begin restoring honesty and integrity to basic biomedical science by rebutting or correcting suspected plagiarism in his Nobel-Prize-winning work.

The centerpiece of this document is an unanswered letter of appeal from the author to Professor Roderick MacKinnon of the Rockefeller University dated November 17, 2003. The aim of the appeal is summarized in the title of this communication. In addition to the 2003 letter, there are also two follow-up letters in this communication, each containing a copy of the 2003 letter and each repeating the appeal. The follow-up letters, dated February 22, 2008 and April 2, 2008 respectively, were also unanswered. To make sure that these letters reached their destination, each was certified with delivery time and date affirmed. Thus the February 22 letter was delivered on the February 24 by the US Postal Service. Two copies of the April 2 follow-up letter were sent. The first copy was delivered by Federal Express on April 4. The second copy of the April 2 letter was delivered by the US Postal Service on the same day. Thus all told three additional copies of the 2003 letters were delivered to, and must be in the hand of Professor MacKinnnon. All these efforts were made to make certain that Professor MacKinnon's refusal to answer my registered 2003 letter was not due to his not having received a copy of that letter.

An ultra simple model of protoplasm to test the theory of its long-range coherence and control so far tested (and affirmed) mostly on intact cell(s).

According to the association-induction hypothesis, the core of living phenomena lies in the long-range, one-on-many connectedness among all three major components of living protoplasm: protein, water and K+ (and the controlling agents, called the cardinal adsorbents.) This article describes simple experimental models that could cogently test the theory of this connectedness and its control by drugs and other cardinal adsorbents.

In response to an open invitation for comments on AAAS project 2061's Benchmark books on science. Part 1: documentation of serious errors in cell biology.

Project 2061 was founded by the American Association for the Advancement of Science (AAAS) to improve secondary school science education. An in-depth study of ten 9 to 12th grade biology textbooks led to the verdict that none conveyed "Big Ideas" that would give coherence and meaning to the profusion of lavishly illustrated isolated details. However, neither the Project report itself nor the Benchmark books put out earlier by the Project carries what deserves the designation of "Big Ideas." Worse, in the two earliest-published Benchmark books, the basic unit of all life forms--the living cell--is described as a soup enclosed by a cell membrane, that determines what can enter or leave the cell. This is astonishing since extensive experimental evidence has unequivocally disproved this idea 60 years ago. A "new" version of the membrane theory brought in to replace the discredited (sieve) version is the pump model--currently taught as established truth in all high-school and college biology textbooks--was also unequivocally disproved 40 years ago. This comment is written partly in response to Bechmark's gracious open invitation for ideas to improve the books and through them, to improve US secondary school science education.

An updated and further developed theory and evidence for the close-contact, one-on-one association of nearly all cell K+ with beta- and gamma-carboxyl groups of intracellular proteins.

The primary focus of this communication is to present an updated and advanced version of the theory of close-contact association of molecules and ions through the spatial fixation and aggregation of the adsorbing sites. The last sections of the text also review a collection of relevant in vitro and in vivo experimental findings gathering since seventy years ago. Though some of these findings were published before the theory, old and new, they all support the theory that close-contact association with the beta-, and gamma-carboxyl groups of intracellular proteins causes the selective accumulation of potassium ions (K+) in living cells.

What befalls the proteins and water in a living cell when the cell dies?

The solvency of solutes of varying molecular size in the intracellular water of freshly-killed Ehrlich carcinoma cells fits the same theoretical curve that describes the solvency of similar solutes in a 36% solution of native bovine hemoglobin--a protein found only in red blood cells and making up 97.3% of the red cell's total intracellular proteins. The merging of the two sets of data confirms the prediction of the AI Hypothesis that key intracellular protein(s) in dying cells undergo(es) a transition from: (1) one in which the polypeptide NHCO groups assume a fully-extended conformation with relatively strong power of polarizing and orienting the bulk-phase water in multilayers; to (2) one in which most of the polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformations (see below for definition) with much weaker power in polarizing-orienting multilayers of bulk-phase water. This concordance of the two sets of data also shows that what we now call native hemoglobin--supposedly denoting hemoglobin found in its natural state in living red blood cells--, in fact, more closely resembles the water-polarizing, and -orienting intracellular proteins in dead cells. Although in the dead Ehrlich carcinoma cells as well as in the 36% solution of native hemoglobin, much of the protein's polypeptide NHCO groups are engaged in alpha-helical and other "introvert" conformation (Perutz 1969; Weissbluth 1974), both systems produce a weak but nonetheless pervasive and "long-range" water polarization and orientation. It is suggested that in both the dead Ehrlich carcinoma ascites cells and in the 36% native bovine hemoglobin solution, enough polypeptide NHCO groups assume the fully-extended conformation to produce the weak but far-reaching multilayer water polarization and orientation observed.

How much water is made "non-free" by 36% native hemoglobin?

At equilibrium, the concentration ratio of poly(ethylene gycol) (PEG-4000) in a dialysis sac containing a 35.1% solution of native bovine hemoglobin over that in the external solution is 0.196 +/- 0.028 (mean +/- SD). This apparent equilibrium distribution constant or rho-value of 0.196, when viewed side-by-side with the near-equal distribution of sucrose and raffinose in similar native-hemoglobin dominated water suggests all (rather than 80%) of the water in this solution has been altered by the native hemoglobin and is no longer free liquid water. Based on Ling's equation for solute exclusion, we found that an excess of water-to-water interaction energy of a mere 4.25 cal/mole could account for both the observed exclusion of PEG-4000 and non-exclusion of sucrose and raffinose. Finally, the long-range action of (even this relatively inactive) native hemoglobin on the dynamic water structure was compared with the exclusion of coated latex microspheres from the altered water 100 microm from the surface of polyvinylalcohol gel (Zheng and Pollack) --in the light of Ling's new theory of ad infinitum water polarization-orientation (under idealized conditions) first publicized at the Gordon Research Conference on "Interfacial Water in Cell Biology" on the campus of the Mount Holyoke College in June 2004.

What determines the normal water content of a living cell?

Most living cells contain a large amount of water. To improve our understanding of this fundamental phenomenon of cell physiology, five theories are critically examined in the light of three sets of relevant experimental findings. These findings are: (1) the diversity and specificity of the percentage water content to tissue type; (2) the limitation imposed by the Law of the Conservation of Energy on postulating membrane pumps and (3) the non-extractability of cell water from the open ends of muscle cells whose membrane covering has been surgically removed. Two of the five theories examined are called respectively the accidental theory (Theory I) and the direct water pump-leak theory (Theory III); both are introduced for the first time here as working hypotheses. Three others theories examined were published; they comprise the Donnan membrane equilibrium theory (Theory II), the indirect pump-leak (Theory IV) and the polarized-oriented multilayer (PM) theory of cell water (Theory V.) The PM (Theory V) alone is in harmony with, and supported by all three sets of the experimental findings. The remaining theories are shown to be non-applicable to cell water by at least two of the findings

A new theoretical foundation for the polarized-oriented multilayer theory of cell water and for inanimate systems demonstrating long-range dynamic structuring of water molecules.

Over the centuries, a vast amount of evidence has been gathering that layers of water sometimes measuring tens of thousands of water molecules thick exhibit altered properties in consequence of exposure to some solid surfaces. Yet, a clear cut theory based on the laws of physics that would predict this kind of long range dynamic ordering of water molecules has been long missing. It is thus with great joy that I announce that a new theory has been developed, which offers theoretical confirmation of the phenomena of long-range dynamic structuring of water by appropriate solid surfaces and which gives clear cut quantitative answers to some key questions about the phenomenon. Thus, for example, under an ideal condition, an idealized checkerboard of alternatingly positively-, and negatively-charged sites of the correct size and distribution could polarize and orient deep layers of water molecules ad infinitum. Based on the quantitative data thus obtained and a relevant simple statistical mechanical law, the new theory predicts that a thin layer of water held between two juxtaposed ideal or near-ideal NP surfaces will not freeze at any (attainable) temperature. On the other hand, water polarized and oriented by an ideal or near-ideal NP-NP system may also not evaporate at temperature hundreds of degrees higher than the normal boiling temperature of water. Both predictions have been confirmed (retroactively) by experimental observations made in the past, accidentally or by design. In a following paper, I will demonstrate that the conclusion reached from the study of the two-dimensional NP surface can be smoothly passed on to the living cells. In the living cell, only one-dimensional linear chains of fully extended protein chains exist. Nonetheless, by proper orientation and distribution, they can achieve similar though less intense water polarization-orientation--as experimentally demonstrated worldwide during the 40 years past.