Research Projects of Gheorghe Benga Outlining the Original Contributions
RESEARCH PROJECTS OF GHEORGHE BENGA OUTLINING THE ORIGINAL CONTRIBUTIONS
My main basic and advanced contributions are in the modern fields of biomedical sciences, namely membrane molecular biology with medical applications and medical genetics.
In the last decades the study of membranes became a top domain of biomedical research, the progress being due to the biochemical methods and physical techniques of molecular biology: X-ray diffraction, electron spin resonance (ESR), nuclear magnetic resonance (NMR) etc. I learned some of these techniques even since I was a Ph.D. student in medical biochemistry at the Department of Biochemistry (lead by Professor I. Manta) of the UMP Cluj, Romania, followed by the post-doctoral specialization in the laboratory of Professor Dennis Chapman (University of London, UK).
The projects of research that I started have been improved following the progress in this field.
To reach the objectives I made special efforts to find adequate means to perform research mainly in Romania. The visits abroad were used to get new means of investigation, to maintain my group in the international stream, to promote the results obtained and to accomplish scientific collaborations of benefit for the Romanian and world science.
- Characterization of molecular composition, structural and functional properties of human liver subcellular membranes.
We described for the first time in the scientific literature the peculiar lipid composition of human liver subcellular fractions (mitochondria and microsomes). The human membranes contain twice more lipids compared with the compared with the rat liver counterparts (i.e. have a higher lipid/protein ratio), the proportion of the lipid classes being the same. There were differences in the composition of essential fatty acids: the human membranes have a higher content of linoleic acid and a lower content of arachidonic acid, a pattern not found in any other mammalian species. Although the unsaturation of membrane lipids is lower in humans (compared to the rat membranes) the fluidity of membranes was found to be higher by spin labeling ESR. This was explained by a weaker immobilization of lipids by proteins in the human membranes. The protein and aminoacid composition of human membranes were also different compared to rat liver, with a higher content of hydrophobic aminoacids. The lipid and protein characteristics of membranes confer to human liver mitochondria pecularities of enzymic activities.
For the first time the fractionation of mitochondria and the separation of the inner and outer mitochondrial membranes, their enzymic and morphological characterization (by electron microscopy) have been achieved for the first time.
The pattern of protein and aminoacid composition of proteins from the liver subcellular membranes are also different in humans compared to rats.
The lipid and protein pecularities of membranes confer to the human mitochondria a greater fragility compared to the rat mitochondria. In addition some enzymic activities have interesting pecularities: ATP-ase, β-hydroxibutiratedehydrogenase and the adenine translocator are stimulated by albumin, while cytochrome oxidase presents a specificity towards oxidation of the human cytochrome c.
The originality and priorities of these findings (see publications in the list below : 9, 12, 15, 18-21, 23, 25, 30, 31, 34, 35, 41, 50, 61, 67, 68, 74, 75, 77, 95, 113, 115, 127) has been acknowledged in a Comment by Prof. Olson, the Editor of « Nutrition Reviews » and citation by Peter Mitchell, Nobel Laureate (FEBS Letters, 151, 147, 1983).
- Interactions between components in biological membranes (in model systems and in natural membranes) studied by spin labeling ESR
a) Protein – lipid interactions
The study of protein-lipid interactions is the fundamental problem of the structure
and function of biological membranes. We studied these interactions in several models and in natural membranes.
Interaction of bovine serum albumin with multilamellar liposomes (model membranes: has been studied especially by spin labeling ESR. I have studied the ESR spectra of spin labels, derivatives of fatty acids, phospholipids or cholestan in the mixture of bovine serum albumin with multilamellar liposomes. It was found that spin labelled fatty acids may not give an accurate picture of protein-phospholipid interactions, because the protein itself can bind fatty acids and spin labelled fatty acids ; hence a partition of spin labels between the protein and lipid can occur. An immobilisation of the fatty acid spin label may only reflect a binding of the labelled molecule to the protein. The use of phospholipid spin labels gives a more accurate picture of protein-phospholipid interactions. In addition to the technical contributions this showed for the first time that free fatty acids can move quickly across lipid bilayer and this may be of significance for the transfer of fatty acids across cell membranes: absorption from the intestine, passage into blood, or into cells in the body (paper no. 24).
As a model of interactions between an intrinsic membrane protein and associated phospholipids we investigated the cytochrome oxidase complex purified from bovine myocardium with spin labels (derivatives of fatty acids and of phospholipids). We showed that phospholipids in contact with the protein (in the so called « boundary layer ») are not completely immobilized (as claimed the scientists who proposed the concept of the « boundary layer »), but may have a relatively high mobility. On the other hand we showed that phospholipids in other layers of the complex could also be influenced by the intrinsic membrane protein (papers nos. 37, 38, 46).
An important paper from the series of those in which I used spin labelling ESR is that in which we studied the binding to serum albumin of the spin label N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide (abbreviated as MSL), which binds covalently to the SH groups in proteins. We found that in previous work of some American authors the interpretation of the spectra of MSL bound to serum albumin was wrong, as the mobile component of the ESR spectrum was due to the marker free in solution (not bound to albumin). We described the conditions under which MSL can be used to correctly investigate the conformational changes of albumin or of other proteins and of the cooperative transitions in mitochondrial membranes (papers nos. 22, 40, 44, 47, 48).
b) Lipid – lipid molecular interactions
A comparison of the effects of cholesterol and 25-hydroxy-cholesterol (25-HC) on the lipid fluidity of liposomes showed that, although 25-HC has smaller effects, it is active at very low concentrations (0,1-5%) at which the fluidity of membranes is influenced (paper no. 57). This might be related with the angiotoxic effects of 25-HC (administered to animals it induces more rapid appearance of the lesions characteristic of atheromatosis). 25-HC results from the oxidation of cholesterol during frying of food or during storage of food for a long time in freezers. Our studies were performed as part of a Co-operative Research Programme (supported by National Science Foundation, USA) between with the group of Prof. F. Kummerow (Burnsides Research Laboratory, Univ. of Illinois, Urbana-Champaign), and the Benga group (Discipline of Cell Biology, UMP Cluj-Napoca).
As part of this research programme we have also studied the effects of the saturation and isomerisation of fatty acids on the red blood cell membranes in rats. These were the first studies in which a standardization of four different kinds of diets administered to rats were performed, such that diets were different only by the degree of saturation and by the isomerisation of fatty acids and not by deficit in essential fatty acids like in the studies of othe authors. Interesting results were obtained, an increased osmotic fragility of red blood cells following incorporation in the membrane (after 4-6 weeks of diet) of trans fatty acids (paper no. 66). As these isomers appear in margarines and hydrogenated oils, such research are important for understanding the molecular mechanisms of appearance of atheromatosis lesions, as well as for the establishment of prophylactic and curative measures in atherosclerosis.
c. Effects of chlorpromazine on biological membranes
We showed that phenotiazines (such as chlorpromazine, CPZ) markedly alters the ESR spectra of erythrocyte membranes labeled with a maleimide spin label (MSL) which binds covalently to the SH groups in membranes. For the first time in the literature we found the origin of spectral changes. These are due neither to conformational changes of the membrane proteins nor to the release of proteins from the membrane (as suggested by other authors). It was demonstrated that CPZ-induced alterations in the ESR spectra are due to a chemical reaction : the reduction by CPZ of the nitroxide free radical. The refects of chlorpromazine in reducing the free radicals in biological membranes were documented by spin labeling ESR and this could have significance in a variety of diseases in which the free radicals are involved (papers nos. 47, 48, 51).
- Studies on the molecular mechanisms of water transport across biological membranes (erythrocytes, liposomes) and medical applications
In 1976 I started a program of research on the study of water transport across biological membranes, aiming to understand the molecular basis, the functional significance and the medical implications of this process. The program brought significant new knowledge, opened new perspectives in the area, inlcuding world priorities, the most important being the discovery of the first water channel protein in the human red blood cell membrane. I am still actively involved in the program, of which I present below the main contribution of the Benga group.
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a. Contributions to the methodology for measuring the water permeability of erythrocytes by NMR
An NMR method for measuring the water diffusion across the red blood cell (RBC) membrane has been evaluated and standardized (papers no. 26, 42, 63). The first paper (26), co-authored with Vasile Morariu, published in 1977 in Biochim. Biophys. Acta, has over 95 citations (approaching the level of 100 citations to become a “Citation Classic”).
b. Caracterization of the water permeability of human erythrocyte
In a series of papers of Benga group in the 1980’s and 1990’s (papers nos. 45, 49, 53, 54, 73) we brought original contributions to the characterization of the diffusional water permeability (Pd), such as: the establishment of the normal values of Pd, describing the pH- and temperature dependence; this made possible the measurement of the activation energy of water transport (Ea,d). These parameters (Pd, Ea,d) have been established for normal subjects of various ages, in the largest series of determinations from the literature (paper no. 106).
We proved that the water transport is achieved by channel proteins, since some SH blocking reagents (SH reagents) inhibit the water transport. We performed for the first time successive incubations with several SH reagents, proving that some of these reagents, such as iodoacetamide (IAM) and N-ethylmaleimide (NEM) do not inhibit the water transport, while the mercury containing reagents, such as HgCl2, p-chloromercuribenzoate (PCMB), p-chloromercuribenzensulphonate (PCMBS) are specific inhibitors, and a maximum level of inhibition exists. For the majority of mercury containing reagents the inhibition could be reversed by cysteine, however we proved for the first time that fluorescein mercuric acetate is an irreversible inhibitor of water diffusion (paper no. 49).
We studied for the first time the water permeability in RBC ghosts and the conditions for inhibition by PCMBS (paper no. 73).
We have also shown that proteolytic digestion of RBC membrane does not inhibit water transport (paper no. 54, the only paper of our group cited by Peter Agre (2003 Nobel Laureate in Chemistry, although in his Nobel Lecture he also refers to our landmark publications, nos. 80, 83).
We studied for the first time the water permeability in RBC ghosts and the conditions for inhibition by PCMBS; we showed that it is necessary an incubation at 370C for at least 5 min to induce significant inhibition (73).
c. Discovery of the first water channel protein in the membrane of the human erythrocyte in 1985, a few years before Peter Agre (2003 Nobel Laureate in Chemistry)
The most important original contribution of my scientific work is the discovery, with my group, in Cluj-Napoca, Romania, for the first time, of the channel protein from the human RBC membrane. This was performed in 1985, and was described in the two landmark papers (nos. 80, 83).
The protein was re-discovered by chance by the group of Agre (Baltimore, USA), who purified the protein in 1988 and found its water channel function only in 1992 (a paper published in Science, in which Agre does not cite our landmark papers. In 1993 the protein was named aquaporin 1.
In 2003 Peter Agre was awarded the Nobel Prize in Chemistry, for “the discovery of water channels”, discovery which in fact was performed by us in Cluj-Napoca in 1985. Our contribution was overlooked by the Nobel Committee. Although in his Nobel Lecture Agre is mentioning me twice (as a “pioneer of the domain of water transport”) he has not cited, even in the printed version of the Lecture, our landmark publications, published in Biochemistry şi Eur. J. Cell Biol. (papers nos. 80, 83).
The recognition of our priority in the discovery of the first WCP is growing: our priority has been acknowledged by many outstanding scientists and has been mentioned in some publications; such publications, as well as opinions of some scientists are presented on the site. The presentation of the discovery is also available on the site.
d. Studies on the purified water channel protein (aquaporin 1)
In 1992 I performed the purification of aquaporin 1 from the human RBC membrane in the laboratory of Prof. John Wrigglesworth (King’s College, Univ. of London) and characterized it by an original procedure of quantitative determination, by densitometry of polyacrylamide gel stained with silver (published in Electrophoresis 17 (4), 715-719, 1996).
In 1994, as “Visiting Professor” at the Department of Protein and Molecular Biology, Royal Free Hospital School of Medicine, Univ. of London (Prof. Dennis Chapman), I performed molecular studies on the structure-function relationships in aquaporin 1 by Fourier-transform infrared spectroscopy. The secondary structure of the protein in its natural medium (phospholipid double layer in water) was investigated and an extremely rapid exchange between the protons in the protein and the deuterium from the heavy water medium was observed (paper no. 129). Such an exchange, in agreement with the channel function of aquaporin 1, was not reported for any other membrane protein.
e. Comparative studies of water permeability of RBCs from various species
In 1989 I started in Romania a programme (prioritaire on the international level) to investigate the water permeability of various species, programme continued in collaboration with the group of Prof. Philip Kuchel (Univ. of Sydney), including including several coworkers (Bob Chapman particularly). We performed several exchange visits. The determinations are performed both at Cluj-Napoca and Sydney. The program is still active, being of great importance for understanding the physiological significance of RBC water permeability.
In 1989 I started in Romania a programme (prioritaire on the international level) to investigate the water permeability of various species, programme continued in collaboration with the group of Prof. Philip Kuchel (Department of Biochemistry, Univ. of Sydney), The determinations are performed both at Cluj-Napoca and Sydney. The program is still active, being of great importance for understanding the physiological significance of RBC water permeability.
We began with determinations of water permeability of RBCs from laboratory animals (mouse, rat, guinea pig, rabbit), continued with domestic animals (sheep, dog, cat, horse, cow), as well as with a variety of wild animals, taking as reference the permeability of the human erythrocytes (papers nos. 116-123, 128, 130, 131, 142, 144, 145, 147, 148, 157, 158, 165, 176, 192, 194, 195). We have now data from over 30 species (paper no. 204).
It is worth mentioning that we described the pecularities of water transport in many Australian species (echidna, platypus, bandicoot, koala, Tasmanian devil, brushtail possum, tree kangaroo, six species of wallabies, Eastern grey kangaroo, red kangaroo, dingo, little penguin, saltwater crocodile), in species introduced to Australia (feral chicken, horse) compared with the European counterpart, as well as in wild species from the Sydney Zoo (elephant, camel, alpaca etc). In addition, we described the protein composition of RBC membranes in these species, the morphological characteristics of RBCs of these species (by light microscopy and electron microscopy, in collaboration with Dr. Guy Cox and Tony Romeo, from the Australian Key Center for Microscopy and Microanalysis, Univ. of Sydney) (papers nos. 116, 165, 194, 195).
We found that there are species (chicken, echidna) in which the Pd values of RBCs are twice lower than in human RBCs and the permeability is not influenced by mercurials ; in the membranes of RBCs of such species there are water channel proteins. In other species (sheep, cow, elephant) the Pd values of RBCs are significantly lower than in human RBCs ; these species are less active than humans. In other species (mouse, rat, guinea pig, rabbit and all marsupianls) the Pd values are higher and the Ea,d values are lower. This indicates a higher role of the water channel proteins in determining the water permeability of RBCs in these species.
Based on these results we proposed a new role for aquaporins in the RBC membranes (papers
nos. 163, 179, 181).
f. Study of changes of water transport in the human red blood cells in pathological conditions (epilepsy, Duchenne muscular dystrophy) and on the genetic determinism of the red blood cell water permeability
We also have the priority in the discovery of the pathological implications of water channel proteins, describing a decreased water permeability of RBCs from children with epilepsy and Duchenne muscular dystrophy. We explained the changes as representing the expression of a genetic generalized membrane defect in these diseases. The paper on epilepsy, result of collaboration with Vasile V. Morariu, my wife Ileana Benga and his wife Cornelia Morariu was published in Nature, 265:636-638, 1977 (paper no. 28). Two papers regarding Duchenne muscular dystrophy, with several co-workers were published in Muscle & Nerve (79, 99). Later other authors confirmed the existence of changes of water transport in these diseases, due to dysfunctions of water channel proteins.
- Studies of ionic changes in epilepsy
In collaboration with my wife and other coworkers we described several changes of electrolytes in plasma, RBCs and cerebrospinal fluid were found in children with epilepsy (29, 39, 55); among these, hypomagnesiemia correlated well with the severity of epilepsy (71).
E. Other research regarding the genetic diseases