Bent Havsteen Curriculum Vitae
Bent Heine Havsteen
Account of Research
H was reared in the shadows of the 2nd World War and during the reconstitution of the European society. He learned the necessity of a good education early and following his interest in the natural sciences, he looked for a university study of international use and recognition. Hence, he chose the study, which was considered as the most difficult in Denmark at the time, because it required the highest marks in the leaving certificate for the admission. That was the chemical engineering study at the Danish Technical University.
After his two years of compulsory service, which were mainly served at the School of Defense against Atomic, Biological and Chemical Warfare, he looked for a position abroad to gain international experience. Since his best foreign language was English, he especially looked for a position in Australia with the help of his uncle, who was the director of an internationally active reinsurance company. That country was chosen because its industrial development was rapidly developing at the time. For that reason, the local need for academic staff was considerable. Since H had written his Masters thesis on the technical biochemical topic (fermentation industry), he applied for a position in the Colonial Sugar Refining Co. Ltd. in Sydney. That company was the fifth largest firm in Australia at the time and employed around 30000 persons.
After an interview in London, H was employed as a research chemist and given the assignment to study the crystallization of sucrose under the direction of Dr. Max Smythe. After a stay in Australia for 13 months, H wished to work for the degree of Ph.D. in Biochemistry. He received the offer to study for the doctorate in colloidal chemistry at the University of Cambridge in England at the cost of the company. However, since this offer carried with it the obligation to return to the company for at least five years, H respectfully declined and preferred to apply for a teaching assistantship at the Department of Biochemistry and Nutrition at Cornell University in the USA with the possibility of gaining the degree of Ph.D.
H also applied for a Fulbright Travel Grant, on which he traveled to the USA. After a year at Cornell, the teaching assistantship was converted into a research assistantship. After the graduation as a Ph.D. in Biochemistry, H was appointed as an Instructor of Biochemistry and as an Acting Principal Investigator at Cornell to sit in for his thesis advisor Professor George P. Hess, who left for a year of Sabbatical Leave in Göttingen. As Professor Hess returned to Cornell, H applied for a NATO science grant for a postdoctoral fellowship at the Max Planck Institute of Physical Chemistry in the Department of Chemical Kinetics under the direction of Dr. Manfred Eigen to continue the research project, which Dr. Hess had begun. After three years of postdoctoral fellowship in Göttingen, H applied for a position as a lecturer and a group leader at the Department of Biochemistry in the Medical School at the University of Aarhus to be able to qualify for the Danish degree of Doctor of Philosophy in the field of Biochemistry. That degree corresponds to the German Habilitation, because it confers the right not only to conduct independent research, but also to teach (jus docendi). Since it was not possible to obtain that degree in Aarhus at the time, it was awarded by the College of Mathematics and Natural Sciences at the University of Copenhagen.
In 1972, H received the encouragement from the University of Kiel to apply for the chair of Physiological Chemistry at the Department of Biochemistry at the School of Medicine, which recently had become vacant upon the retirement of Professor Hans Netter. H participated in the contest for the chair and received the offer to succeed Professor Netter in October 1972.
Research Field Enzyme Mechanism
The main research topic during the entire scientific career of H was the mechanism of enzyme catalysis. This topic has fascinated him ever since he did his first research work during his compulsory service at the Royal Danish School of Pharmacy on the inhibition of acetylcholine esterase by organophosphates. This interest was reinforced during his work on the crystallization of sucrose in Sydney, since he discovered that the inhibition of the growth centers (dislocations) on the sucrose crystals by oligosaccharides like kestose could be described by the terminology of enzyme kinetics.
At Cornell, the main topic of research was the mechanism of chymotryptic catalysis, since this digestive enzyme had been studied intensively already. and since it was cheap and available in large amounts. We inhibited this enzyme with an organophosphate and with haloketones, which H synthesized himself. Subsequently, the structural differences between the free and the inhibited enzyme were characterized by UV-spectrophotometry, optical rotatory dispersion and acidimetric titration curves. H found that 2 of the 4 tyrosine residues had suffered a large displacement of their pK-values by the specific acylation of the active site, which indicated a condensation of the structure in the environment of the active site. This hypothesis was sustained by the measurement of a change in the secondary structure. Later, that conclusion was definitely confirmed by x-ray diffraction studies.
The main thesis of H for the doctorate was the existence of a conformational change in chymotrypsin accompanying its catalysis, which regulated its activity. The titrations curves had shown that an ionizing group of a pK-value around 9 had caused the conformational change by a pK-displacement induced by the binding of the specific substrate. This group was identified tentatively by its pK-value and the total inactivation of the enzyme by specific acetylation of the α-amino groups as an N-terminus. The complete elucidation of the spatial structure of the enzyme revealed later that this group belonged to the N-terminus of the B-chain of the enzyme. It functioned as the switch of the enzymatic activity.
In Göttingen, where Drs. Manfred Eigen and Louis DeMaier had developed the chemical relaxation techniques, with which studies of the elementary steps of fast chemical reactions were possible, Professor Hess had begun a project on the interaction of chymotrypsin with the chromogenic, competitive inhibitor proflavine. It was continued by H. The results showed beyond doubt that the initial binding of the substrate to the enzyme was followed by an isomerization of the enzyme-inhibitor complex. This confirmed the hypothesis, which had been forwarded at Cornell.
At Göttingen, H investigated the possibility that this mechanism were general. He was able to demonstrate another example using the glyceraldehyde-3-phosphate dehydrogenase, which is a key enzyme in the glycolytic pathway. Besides, he found a conformational change in cytochome C, which is a protein in the respiratory chain. This conformational change accompanied the electron transfer. In Göttingen H, also build a new temperature jump apparatus in cooperation with Dr. DeMaier. With this instrument, it was possible to study short-lived inter-mediate compounds by applying temperature jumps to solutions in movement, even when the available quantities of substance were low.
In 1965, H accepted a position as a lecturer and a group leader in Biochemistry at the Institute of Biochemistry of the School of Medicine at the University of Aarhus to be able to prove that he was capable of conducting independent research. Since the spatial structure of chymotrypsin had been elucidated recently at Cambridge, H build a 3-dimensional model of this enzyme in the scale of 1: 50 x 106 (ca. 2 x 2 x 2 m). On this model, he could measure the distances between the chemically modifiable functional groups directly. Subsequently, he formed covalent bridges across the loop that participated in the activity-regulating conformational change using bifunctional imidates in the hope to gain an enzyme of higher specific activity, which did not autolyze. In this way, he succeeded in preparing a very stable enzyme, which possessed a thousand times higher specific activity than that of the native form. H then reduced the molecular mass of the modified enzyme by limited proteolysis. It could be cut down to about 20 % of its original size without loosing much of its activity and it had become much less immunogenic. The latter property is of importance by clinical applications.
Another project, which H began in Aarhus, was the study of enzymatic activity in solvents of low water content. The idea behind this project, which originated in Switzerland, was to render peptide synthesis in a water-arm solvent using proteolytic enzymes possible. In this way, H succeeded in preparing oligopeptides of amino acids with hydrophobic sidechains using dioxane-water mixtures. This method was soon applied to humanize pig insulin industrially, since the pig variety of this hormone only differs from the human form in the C-terminal amino acid.
H was 1968 promoted to the degree of Doctor of Philosophy in the field of Biochemistry at the School of Mathematics and Natural Sciences at the University of Copenhagen. His thesis was a generalization of the idea of conformational changes accompanying enzyme catalysis. This thesis has remained accepted in the following 35 years. When H 1972 received the call on the 2nd chair of Physiological Chemistry at the Medical School of the University of Kiel, he dissolved his group in Aarhus and formed a new research group in Kiel: It was joined by two of his former acquaintances from Göttingen. He moved into the 1st floor of the building, which had been vacated by the Institute of Physiology recently, when the latter moved into its newly built premises. The old laboratories were completely renovated during the following year and furnished with new furniture and instruments. A modernization was not deemed necessary until his emeritation 29 years later.
In Kiel, H continued his studies of enzyme mechanisms. He developed a method to the complete elucidation of the kinetics of any case of enzyme catalysis in solution. One result of this was that Dr. Ramós Varon from the University of La-Mancha in Albacete offered the assistance of his group in the study of further, complicated biochemical reactions mechanisms. About fifty scientific articles in highly rated, peer-reviewed journals have since been the result of this co-operation.
The study of the proteases was extended to enzymes in the digestive juice of the carnivorous plant Nephendes on the recommendation of the microbiologists of the university. It turned out that both the structural and the functional properties of these enzymes very much resembled those of their human counterparts.
The Kinetics of Osmosis
Since the chair of Professor Hans Netter was known world-wide for its work on biological membranes, and since these structures had not been sufficiently investigated at the time in spite of their importance and actuality in connection with the studies of active transport phenomena, H decided to begin a project on membranes. He remember from his studies in Copenhagen a comment by Professor Brĝndsted that the osmotic power, which was evident in plants and animals, could be used to generate energy, if suitable artificial membranes could be prepared. In that case, the difference in the concentration of salt in ocean and river water would suffice to operate a major energy plant.
H had noticed that no research work had aimed at the elucidation of the kinetics of osmotic flow, although the osmotic power since the days of Ostwald had been studied intensively. Recently, several new, artificial membranes had been prepared on the basis of cellulose acetate and polycarbonate, which would be suitable for such studies due to their small pore widths (ca. 30 Ċ). Hence, H built a small cell to house such a membrane, which was available from the Membrane Division of the Danish Sugar Factories. On one side of the membrane, a salt solution of known concentration was added and a capillary tube, through which the osmotically drawn water could rise, was introduced. On the other side of the membrane distilled water was placed. The concentration dependence of the osmotic flux velocity was measured, and the associated kinetic parameters were calculated. Then, an equation could be formulated, with which the osmotic flux could be calculated from the atomic parameters of the ions present.
Unfortunately, a calculation of the power generated by a large-scale plant showed that such a process was not economically feasible currently due to the high costs of the membranes and their maintenance. However, today, 20 years later, the situation is different, because new and more efficient membranes have been developed in the mean time. Hence, the Danish Technical University will conduct large-scale tests in Norway this summer to ascertain the feasibility of the operation of a modern osmotic power plant. Another possible application of the equation mentioned above, which also is applicable to organic compounds, is the calculation of the dialysis time required to purify the blood of kidney patients. Since a long dialysis period increases the risk of hemolysis, such an application could be beneficial to the patients.
Oxazolones
A number of amino acid derivatives called oxazolones were synthesized in cooperation with the assistant Dr. rer.nat. Hans-Jürgen Baese and tested for their suitability as substrates for proteases in the blood. Dr. Baese prepared a large number of chromogenic and fluorescent oxazolones, which were used for the determination of the operational normality of the active site and the specific activity of serum serine proteinases. These enzymes appear in the case of blood coagulation and leukemic diseases. Since the enzyme catalyzed opening of the oxazolone ring is accompanied by a large displacement of the absorption and the fluorescence emission spectra, these substances are sensitive reagents for the tests of the hemostatic state. This project, which was the basis of the thesis of Dr. Baese for the medical doctorate, was received by the hematological laboratory of the Clinic of Internal Medicine with satisfaction.
Participation in Research Program 11
The lymphatic System and experimental Transplantation
When the research group of H was reinforced by several assistants with experience in the biochemical immunology, a number of projects were submitted by the chair to the board of the research group for consideration as integral parts of the program. One of these projects, which was conducted by Dr. Han Bauman from the University of Leiden characterized structural changes in the plasma membrane of lymphocytes that had suffered an oncogenic transformation. By some types of leukemia, we found a direct proportionality between the content of sialic acid on the outside of the plasma membrane and the index of malignity, i.e. the stage of the development of the disease. This conclusion was primarily supported by measurement of the electrophoretic mobility of isolated plasma membranes, since each molecule of sialic acid carries a negatively charged carboxyl group. This method has the advantage of requiring only a short time.
Another research project, which was proposed in cooperation with the assistants Dr. Hilmar Lemke, Dr. Hinrich Hansen, Dr. Peter Froese and Dr. Hannes Gerdes, was the preparation of monoclonal antibodies against cell surface epitopes, which are characteristic for leukemic lymphocytes. Two such MIg, Ki-1 and Ki-67, proved to be particularly useful, because Ki-1 could be used for a rapid and reliable detection of Morbus Hodgkin cells, whereas Ki-67 could be used for the detection of an intracellular epitope that indicates a rapid cell proliferation, which is typical of many cancer diseases.
Since several projects from the chair were accepted by the research program SFB 111, and since a representative of the field of biochemistry was welcome, H was elected to the board of this program. When the SFB 111 was prolonged after the first period of operation, an additional project from the chair was incorporated in the research program. It was a further development of the Ki-1 project, which aimed at the definition of the signal pathways of leukemic cells from the plasma membrane through the cytoplasma to the cell nucleus. Two epitopes were recognized by Ki-1. One of them was bound to the plasma membrane. After the analysis of the amino acid sequence, it became apparent that this molecule was a tumor necrosis factor receptor, which had been inactivated by mutation. Hence its function as a sensor on the outside of the plasma membrane had been abolished. The other epitope was soluble. It was phosphorylated by a serine/threonine-specific protein phosphokinase and passed the nuclear pore to approach the RNA-producing nucleosomes. Unfortunately, the amino acid sequence of the latter epitope could not be completely elucidated because of the existence of particularly stable secondary structures in its RNA. We found evidence of a close relationship between this epitope and the regulatory protein τβ, but a closer investigation of this interaction is still in progress.
Fetal immunological Impression through the Plasma Membrane
An extension of one of the SFB-projects, which was supervised by Professor Hilmar Lemke, was his idea that immune networks, such as those proposed by the nobel laureate Niels Jerne, played a role in the communication between immune cells. Dr. Lemke had observed that immunological properties of mice could be passed on from generation to generation in a non-genetic fashion. He confirmed this by immunizing pregnant mice shortly before the birth with a non-physiological, synthetic antigen and analyzing the immune response in the new-born mice. Since a genetic response requires a longer period of time than the interval between the immunization of the mother and the birth, a genetic mechanism could be excluded. A direct transfer of immunoglobulins across the placenta membrane could also be excluded. Dr. Lemke is still trying to find an explanation of this phenomenon.
Isolation and Characterisation of the ACTH-Receptor
The biologist Hartmut Lüddens applied in the early 1980ies for working space in our laboratory to work for his doctorate on an endocrinological subject. Since Dr. Hilmar Lemke shortly before had joined our group with the initial goal to establish the technique of preparation of monoclonal antibodies, and since the hormone producer Ferring Corp. had transferred its main research department to Kiel recently, I suggested a cooperation between Dr. Lemke, Ferring and Hartmut Lüddens. Important reasons were that Dr. Lemke was the first German scientist, who had learned the technique, that Ferring already possessed much endo-crinological experience and that the graduate student had a strong interest in applying monoclonal antibodies to the investigation of hormone mechanisms. That field of endeavour had been neglected so far.
We gave Hartmut Lüddens the research project in cooperation with Dr. Lemke to establish monoclonal antibodies against surface epitopes on porcine, cortical adrenal cells. The pig was chosen as a model, since the biological material needed could be acquired easily and cheaply from the local slaughterhouse. Among the monoclonal antibodies, which were prepared, two were of special interest. One of them inhibited the synthesis of steroid hormones in the adrenal gland and the other mimicked the action of ACTH (corticotropin), i,d, it stimulated the production of steroid hormones. Hence, in the case of pathological disturbances of the adrenal function, we should be able to counteract. A binding analysis according to Scatchard showed that two ACTH-receptors existed on the outer surface of the plasma membrane of the porcine, cortical, adrenal cells and that their binding affinity widely differed.
We were also able to show that these receptors were coupled to the GTP-metabolism, which later has been confirmed by other workers. One of the ACTH-R-specific antibodies was covalently linked to the ACTH-receptor using an UV-light-sensitive cross-linking reagent, and the complex was isolated. The molecular mass of the receptor was then determined by SDS-polyacrylamide electrophoresis. Unfortunately, the gene technological techniques were not yet available at the time, so it was not possible to determine the primary structure of the ACTH-receptor. Hartmut Lüddens received high honors for his doctoral dissertation, and he won the first installment of the Ferring Price, which was the highest price offered in Germany at the time. He left Kiel soon to work at the NIH in the USA. A few years later, he continued his successful scientific career at the European Institute of Molecular Biology in Heidelberg, until he received an offer of a chair in Molecular Endocrinology at the University of Mainz in Germany.
Cooperation with the Group of Professor Busse
H was acquainted with Dr. Busse already in Göttingen, as the latter worked on his doctorate with Professor Manfred Eigen. Dr. Busse joined the chair around 1975. His line of research was the non-linear relationships in biochemistry. He began with pure model systems like the Belousou-Zhabotinski-reaction that simulated part of the citrate cycle. Later, he extended his work to work on biological rhythms, glycolytic relaxation oscillations and pattern generation of fungi. Later, he learned gene technological techniques during a stay in the laboratory of Professor Eisner at the Harvard Medical School. He applied these methods to the determination of the nucleotide sequence of the developmentally important HOX-genes of the fruit fly Drosophila melanogaster and the domestic chicken.
He continued his developmental studies with an automatization of the microscopic video-registration of the organogenesis of the nematode Chenorhabditis elegans. Another project, which he began, was the regulation of the neurite formation by plasma membrane-bound receptors. An Indian postdoctoral fellow participated in this project by the preparation of membranes, which were soaked in the insecticide pyrethroide and placed in defined positions in the insect brain. Subsequently, the dendrite formation was inhibited locally. The technique of placing insecticide-soaked membranes of the size of 20 x 20 cm in banana and cotton fields is considered as a standard economical, technique that has considerable advantages in the conservation of nature. The method, which is called controlled release, prevents the usual losses of up to 50 % due to insect attacks.
Clinical Research Requests to the Chair
The accept of a chair at a Medical School in Germany is associated with responsibilities, which exceed those of teaching, basic research and administration that are shared with the colleagues in Schools of Science, because it is expected that the chair holder places all his resources at disposal in the case of a clinical crisis that cannot be controlled by routine techniques. H has always welcomed such tasks, because he found them interesting and gave him an opportunity to show that his presence in the Medical School was appropriate. H was grateful for this sign of confidence from his clinical colleagues. Around 1980, the first of these assignment appeared.
Ethanolaminosis
A consultant from the Pediatric Clinic approached H to solicit his help, because he had received two patients from the same family, who probably suffered from a new, inborn disease, which could be treated symptomatically only, since the pathogenesis was unknown. The symptoms of the disease did not appear, until the age of several months was reached, at which time both the physical and the psychological development almost ceased. Particularly the liver, the heart and the spleen hypertrophied, and the signal transmission from the brain to the muscles was interrupted to a large extent. The children died from heart failure in an age of about 18 months. Since the enlargement of the organs indicated the existence of a genetic storage disease, H received for analysis a small liver sample that had been removed during the autopsy. A considerable cell enlargement was visible in the microscope, and ninhydrin-positive conglomerates, which were not enclosed by membranes were accumulated in the cytoplasma.
We isolated the unknown substance, which seemed to be rather homogenous. It had a small molecular mass and reacted not only with ninhydrin, which signaled the presence of an amino group, but also with the PAS-reagent (periodate-aldehyde-sulphite). Hence, two groups in neighbor position were amino or alcohol groups. Since the substance was not a carbohydrate and moved chromato-graphically as ethanolamine, we identified the substance as ethanolamine. Subsequently, we determined the specific activity in the autopsy sample of the enzyme ethanolamine kinase, which is preparing ethanolamine for its incorporation in the cellular membranes, particularly the plasma membrane, by phosphorylation. Since this is also the case in neurons, we could explain the neurological failures with the formation of faulty synaptic membranes.
The specific activity of the ethanolamine kinase in the patient sample was about 15 % of the normal value for a child of that age only. Hence, the responsible structural gene of the enzyme had probably been damaged by mutation. Unfortunately, the modern gene technological methods were not yet available. Therefore, we were not able to identify the mutation and determine the primary structure of the enzyme. Hence, an exhaustive analysis of the disease must be left in the hands of the next generation of biochemists. Shortly after the publication of our work on the new inborn error, a third child presenting the same symptoms was discovered in Oslo, Norway, but we were not able to obtain any pathological samples. Unfortunately, this child also died, since the methods of gene therapy were too new to warrant practical applications. Since the Pediatric Clinic informed us that about half of the children, who died early in the developed countries, were buried without a diagnosis, it is possible that the new inborn error is more widespread than it appears now.
A Coagulopathy acquired by lay Treatment
Shortly following the conclusion of the case mentioned above, H received a call on the telephone from a consultant from the university hospital in Lübeck, which is situated about 100 miles East of Kiel. He asked for help from the biochemists in the diagnosis and the development of a treatment of a patient suffering from an unknown, acquired disease. The patient suffered from a prostate adenoma, which had to be operated upon within two weeks, because his urine had to be removed daily through the pelvic wall with a syringe under the risk of infection. Unfortunately, this patient could not be subjected to surgery, because his blood could not coagulate. He had acquired the latter disease by a self medication. Since he feared a cancerous disease, he had taken intramuscular injections of a raw plant extract that had been recommended to him from lay practician. Unfortunately, this extract contained a substance, which was strongly immunogenic. It released the formation of antibodies against an epitope that was present on one or more of the coagulation proteins also. The consultant gave H a time limit of 14 days to solve the problem.
Since coagulation proteins like other blood proteins are conjugated with carbohydrate chains, H guessed that one of these harbored the fatal epitope, the blockage of which was responsible for the failure of the coagulation of the blood. Hence, we removed the carbohydrate chains from the patient blood sample, which we had received. Following this treatment, the patent blood was able to coagulate again. Since the monosaccharide fucose that is ubiquitous to human tissues only can be placed in the terminal position of the oligosaccharide chains, H guessed that this sugar contained the epitope sought. We confirmed the correctness of this hypothesis by binding the fucose to dextrane gel covalently. This modified gel was poured into a chromatographic column, and the blood sample of the patient was passed through this column. Since the eluate was able to coagulate now, we had proved that the antibody that was responsible of the coagulopathy had been removed. Now, we eluted the substance, which had been bound to the column, and showed that it was an antibody. It was able to inhibit the coagulation of normal blood.
After the completion of the proof of our hypothesis, the task remained to design a suitable procedure, with which the patient could be treated. We recommended the oral intake of a large dose of the harmless, physiological compound fucose just before the operation to displace the antibody from the coagulation factor. Since the toxicologist in Lübeck had no objections, the patient was treated in the way and received the necessary surgery, which was successful and did not give rise to any vascular crisis. However, since the epitope was present in all joints also, where the antibody, which the patient would produce for the rest of his life, caused pain, he would have to take a spoonful of fucose, each time his pains became intolerable. We even managed to solve the problem within the time limit.
Porcine Replacement Heart Valves
Around 1990, our heart surgeons called our attention to the problem that the porcine heart valves, which they had been using to replace defective human valves, in the course of a few years hardened and failed to close the artery completely. Besides, pieces from the edges of the replacement valves fell off and caused vascular disturbances. Hence, they had to be replaced again. A general agreement existed about the cause of this problem. It was ascribed to the method of conservation of the native porcine valves, which aimed at a reduction of its immunogenity and a reinforcement of the structure. Our assignment was therefore to design an improved method of conservation.
The current procedure was to open the pig heart along a horizontal plane and to pour a cooled solution of succin-dialdehyde directly onto the valve to cross-link collagen. After the period necessary for the reaction, the valve was cut out and stored at -20˚ C. The problem by the use of the dialdehydes was that they could not be prepared in the pure form. Therefore, H together with a graduate student tried other cross-linking reagents, which had served well on several other proteins, especially diimidates. However, all the alternative reagents introduced a new problem, because they were all associated with a high heat of reaction, which locally denatured the valve. Hence, H had to admit that he had encountered a problem, which he could not solve. This time he had to acknowledge that he arrived at the limit of his capability. In stead, he constructed a new instrument to measure the flexibility of native and chemically modified heart valves, with which the graduate student successfully completed his work for the medical doctorate.
Mamma Carcinoma Samples of German and Swedish Women
Around 1980, the German pathologist Dr. Steinbeck and his wife asked H for scientific support for the analysis of about 50 mamma carcinoma samples, which they and the German gynacologist Dr. Arp, who worked in Sweden, had removed by biopsy. Since Dr. Busse and his group together with Dr. Han Bauman from the SFB 111 had established the two-dimensional SDS-polyacrylamide-electrophoresis in our laboratory, primarily for the analysis of numerous lymphoma samples, Mrs. Steinbeck was appointed as a graduate student and given the task of analyzing the mamma carcinoma biopsies using the SDS-PAGE-technique.
They worked on the large material using the traditional 20 x 20 cm large gel plates, on which they carried out an electrofocussation in the first dimension and an SDS-polyacrylamide electrophoresis in the second. The protein patterns, which had been made visible by staining with amido-black and silver was calibrated with known proteins that served as markers. Direct comparisons between the patterns permitted a classification that was correlated with clinical criteria, e.g. the presence or absence of hormone receptors. However, because of the large volume of the plates, we decided in cooperation with Dr. Joachim Das to miniaturize the technique. A new electrophoresis apparatus, in which a gel plate of the size 5 x 5 cm could be placed, was constructed by the workshop of the Institute. These gel plates could be enclosed in the slide frames and projected on the wall in large scale. In this way, the resolution of the method was enlarged ten fold. Consequently, many proteins, which previously had been invisible, could be detected and registered.
The analysis of such protein patterns had been performed by Dr. Klas Westerbrink from the SFB 111 already. He refined the procedure be introducing topological methods. Our pathologists decided to transfer this technique to the analysis of the mamma carcinoma biopsies. However, at first they preferred to prepare the results they already had gained for publication. Hence, the many gel slides were stored for an analysis later. The preliminary presentation of the data in the form of a publication was successful, and the results were received by the colleagues with satisfaction. Unfortunately, the slides with the data were discarded during a modernization of the laboratory by strangers during a vacation, during which Dr. Busse and his group were absent. Therefore, the entire project must be repeated as soon as possible.
Scientific Assignments from the University
Cooperation with Professor Jingui Shao, Hangzhou
The University has a number of partnerships with foreign universities, e.g. the Medical University of Zhekiang at the old, Chinese empirial town of Hangzhou.
The Department of Foreign Relations of the university administration requested the assistance of H to encourage the modernization of the biochemical research in Hangzhou. Professor Shao appeared in Kiel and presented H with a list of wishes for cooperation on research projects. One of these was the identification of an anti-cobravenom antitoxin, which Professor Shao had discovered in the blood of the Chinese cobra snake by electrophoresis. We isolated this antitoxin, which was a protein. A determination of the amino acid sequence of this protein showed that this protein was the serum albumin that in addition to the usual properties of a serum albumin had acquired the ability per molecule with high affinity to bind up to 6 molecules of the cardiac toxin, which is the most dangerous component of copra venom. We determined the accessibility of the toxin binding sites by a kinetic analysis, in which a membrane with the antitoxin attached was percolated with a solution containing the cobra toxin.
The other project, which we worked on, was the lactose intolerance that in widespread is East-Asia. Our contribution to the solution of this problem was to provide a gene technologically modified bacterial culture that overproduced a lactase of high specific activity. This cheap enzyme, which could be isolated by filtration, could be added directly to the milk. Subsequently, it cleaved the lactose to the harmless monosaccharides galactose and glucose. H paid Professor Shao a visit in the early 1990ies to read seven lectures to the students and staff in Hangzhou and Shanghai on biochemical topics of high actuality.
Cooperation with Professor Yu, Jian
Professor Yu of the Institute of Traditional Chinese Pharmacology and Medicine in another empirical city, Jian, had isolated an anti-cancer drug from a plant resembling cucumber. This compound was a terpene named tubeimosine, which Professor Yu had used with success to treat large tumors. He knew our work in the SFB 111 and solicited now our help to elucidate the mechanism of action of tubeimosine. Since the molecular structure of this substance resembled known affectors of protein kinase C, and since this enzyme plays an important role in many oncogenic processes, H suggested that tubeimosine interacted with protein kinase C. Hence, we developed an assay for this enzyme, and H prepared in addition a sketch project of a factory, which could produce sufficient amounts of tubeimosine for the high demand in China. Around 1995, H visited the Institute of Traditional Chinese Pharmacology and Medicine at Jian to introduce the methods and to give a series of five lectures on biochemical topics of special interest to the students and staff at Jian.
Cooperation with the University of Tartu, Estland
The University of Kiel received a request from the Commissariat of Research of the EU-Commission to participate in a Joint European Project in cooperation with the universities of Lübeck, Stockholm and Turku with the purpose of modernizing the teaching and research in biochemistry at the only estic university, the one at Tartu. This university, which formerly was known under the name of Dorpat, was erected about 500 years ago by German noblemen to serve as an educational institution for their children. The university administration at Kiel gave H the assignment to coordinate this project as a local director. A group of five scientists from our department paid Tartu two visits each for two weeks to read 2 x 10 lectures on biochemical topics.
Two groups of about six advanced biochemistry students from Tartu spent two periods of each four months to participate in all advanced laboratory courses and to take part in the research project, which currently were in progress in Kiel. Besides, we supplemented the biochemistry library in Tartu with many modern textbooks on this and similar topics. Besides, about ten biochemistry teachers from Tartu visited Kiel twice to gain inspiration and advice for their future work. The EU rated our work for the Joint European Project as very satisfactory.
Flavonoids
The origin of this project was an irritation, which H felt, when he received several brochures, in which a bees wax named Propolis was praised in an irresponsible way for its many curative effects on widespread diseases. H approached the producer and accused him for irresponsibility in the propagation of unproved statements on propolis. Since this bee-keeper reacted sensibly upon the serious allegations, H accepted his offer to supply further quasi-scientific evidence of the effects of propolis for a more thorough examination. The material, which mainly originated from Eastern European sources, supported some of the claims made, but the methods that had been used were outdated and lacked controls. Since it would be unreasonable to expect that a bee-keeper could judge scientific evidence correctly, H accepted the offer to receive a sample of propolis for his own experiments.
Chromatographic analyses showed that propolis contained appreciable amounts of the plant pigments named flavonoids. Hence, H read the scientific literature on the flavonoids in journals of high rank. He found a large number of articles from highly recognized institutions and reviewed this material. More than 1400 scientific articles of high quality were evaluated, and the conclusion that the flavonoids actually possessed properties, which under suitable conditions could have a positive effect on human health and the course of diseases. This conclusion was published in two review articles, which appeared in 1983 and 2002. Both of them were quoted frequently. In the course of two decades H received many samples of propolis from most parts of the world. He analyzed them chromatographically and found that they on the average contained about 25 different flavonoids in appreciable concentrations. These could be applied for medical purposes, if the conditions were suitable.
Summary
In his scientific career, H has always tried to give his staff the best possible framework for their work. Besides, he has made every effort to establish bridges