Kluyver, Albert Jan WORLD OF MICROBIOLOGY AND IMMUNOLOGY 325 • • nine tRNA. The nucleotide sequence of this tRNA had been determined in Robert Holley’s laboratory. In 1970, when Khorana announced the total synthesis of the first wholly arti- ficial gene, his achievement was honored as a major landmark in molecular biology. Six years later, Khorana and his associ- ates synthesized another gene. In the 1980s, Khorana carried out studies of the chemistry and molecular biology of the gene for rhodopsin, a protein involved in vision. In 1966, Khorana was elected to the National Academy of Sciences. His many honors and awards include the Merck Award from the Chemical Institute of Canada, the Dannie- Heinneman Prize, the American Chemical Society Award for Creative Work in Synthetic Organic Chemistry, the Lasker Foundation Award for Basic Medical Research, the Padma Vibhushan Presidential Award, the Ellis Island Medal of Honor, the National Medal of Science, and the Paul Kayser International Award of Merit in Retina Research. He holds Honorary Degrees for numerous universities, including Simon Fraser University, Vancouver, Canada; University of Liverpool, England; University of Punjab, India; University of Delhi, India; Calcutta University, India; University of Chicago; and University of British Columbia, Vancouver, Canada. See also Genetic regulation of eukaryotic cells; Microbial genetics KITASATO, SHIBASABURO (1852-1931) Kitasato, Shibasaburo Japanese bacteriologist Bacteriologist Shibasaburo Kitasato made several important contributions to the understanding of human disease and how the body fights off infection. He also discovered the bacterium that causes bubonic plague. Born in Kumamoto, Japan, Kitasato, completed his med- ical studies at the University of Tokyo in 1883. Shortly after, he traveled to Berlin to work in the laboratory of Robert Koch. Among his greatest accomplishments, Kitasato discovered a way of growing a pure culture of tetanus bacillus using anaer- obic methods in 1889. In the following year, Kitasato and German microbiologist Emil von Behring reported on the dis- covery of tetanus and diphtheria antitoxin. They found that ani- mals injected with the microbes that cause tetanus or diphtheria produced substances in their blood, called antitoxins, which neutralized the toxins produced by the microbes. Furthermore, these antitoxins could be injected into healthy animals, provid- ing them with immunity to the microbes. This was a major find- ing in explaining the workings of the immune system. Kitasato went on to discover anthrax antitoxin as well. In 1892, Kitasato returned to Tokyo and founded his own laboratory. Seven years later, the laboratory was taken over by the Japanese government, and Kitasato was appointed its director. When the laboratory was consolidated with the University of Tokyo, however, Kitasato resigned and founded the Kitasato Institute. During an outbreak of the bubonic plague in Hong Kong in 1894, Kitasato was sent by the Japanese government to research the disease. He isolated the bacterium that caused the plague. (Alexandre Yersin, 1863 – 1943, independently announced the discovery of the organism at the same time). Four years later, Kitasato and his student Kigoshi Shiga were able to isolate and describe the organism that caused one form of dysentery. Kitasato was named the first president of the Japanese Medical Association in 1923, and was made a baron by the Emperor in 1924. He died in Japan in 1931. See also Antibody and antigen; Bacteria and bacterial infec- tion; Immunity, active, passive and delayed; Immunization KLUYVER, ALBERT JAN (1888-1956) Kluyver, Albert Jan Dutch microbiologist, biochemist, and botanist Albert Jan Kluyver developed the first general model of cell metabolism in both aerobic and anaerobic microorganisms, based on the transfer of hydrogen atoms. He was a major exponent of the “Delft School” of classical microbiology in the tradition of Antoni van Leeuwenhoek (1632–1723). Outside Delft, he also drew on the legacy of Louis Pasteur (1822–1895), Robert Koch (1843–1910), and Sergei Nikolayevich Winogradsky (1856–1953). Born in Breda, the Netherlands, on June 3, 1888, Kluyver was the son of a mathematician and engineer, Jan Cornelis Kluyver, and his wife, Marie, née Honingh. In 1910, he received his bachelor’s degree in chemical engineering from the Delft University of Technology, but immediately shifted his focus toward botany and biochemistry, winning his doctorate in 1914 with a dissertation on the determinations of biochemical sugars under the tutelage of Gijsebertus van Iterson, professor of microscopic anatomy. In 1916, on van Iterson’s recommendation, the Dutch government appointed Kluyver as an agricultural and biological consultant for the Dutch East Indies colonial administration. In 1921, again on van Iterson’s recommendation, Kluyver succeeded Martinus Willem Beijerinck (1851–1931) as director of the microbiology laboratory at Delft, where he spent the rest of his career. He immediately acquired the most modern equipment and established high standards for both collegiality and research. The reorganized laboratory thrived. Kluyver’s reputation soon attracted many excellent graduate students, such as Cornelius Bernardus van Niel (1897–1985), another chemical engineer. Van Niel received his doctorate under Kluyver with a dissertation on propionic acid bacteria in 1928 and was immediately offered an appointment at Stanford University. In a landmark paper, “Eenheid en verscheidenheid in de stofwisseling der microben” [Unity and diversity in the metab- olism of microorganisms] Chemische Weekblad, Kluyver examined the metabolic processes of oxidation and fermenta- tion to conclude that, without bacteria and other microbes, all life would be impossible. Two years later he co-authored with his assistant, Hendrick Jean Louis Donker, another important paper, “Die Einheit in der Biochemie” [Unity in biochemistry] Chemie der Zelle und Gewebe, which asserted that all life forms are chemically interdependent because of their shared womi_K 5/6/03 3:29 PM Page 325 Koch, Robert WORLD OF MICROBIOLOGY AND IMMUNOLOGY 326 • • and symbiotic metabolic needs. He explained these findings further in The Chemical Activities of Microorganisms. Kluyver had a knack for bringing out the best in his stu- dents. He often and fruitfully collaborated and co-published with them, maintaining professional relationships with them long after they left Delft. For example, with van Niel he co- wrote The Microbe’s Contribution to Biology. A cheerful, friendly, popular man, he was widely and fondly eulogized when he died in Delft on May 14, 1956. Van Niel called him “The Father of Comparative Biochemistry.” See also Aerobes; Anaerobes and anaerobic infections; Azotobacter; Bacteria and bacterial infection; Biolumines- cence; Escherichia coli (E.coli); Microbial symbiosis; Microbial taxonomy; Microscope and microscopy; Yeast KOCH, ROBERT (1843-1910) Koch, Robert German physician Robert Koch pioneered principles and techniques in studying bacteria and discovered the specific agents that cause tuber- culosis, cholera, and anthrax. For this he is often regarded as a founder of microbiology and public health, aiding legislation and changing prevailing attitudes about hygiene to prevent the spread of various infectious diseases. For his work on tuber- culosis, he was awarded the Nobel Prize in 1905. Robert Heinrich Hermann Koch was born in a small town near Klausthal, Hanover, Germany, to Hermann Koch, an administrator in the local mines, and Mathilde Julie Henriette Biewend, a daughter of a mine inspector. The Kochs had thirteen children, two of whom died in infancy. Robert was the third son. Both parents were industrious and ambi- tious. Robert’s father rose in the ranks of the mining industry, becoming the overseer of all the local mines. His mother passed her love of nature on to Robert who, at an early age, collected various plants and insects. Before starting primary school in 1848, Robert taught himself to read and write. At the top of his class during his early school years, he had to repeat his final year. Nevertheless, he graduated in 1862 with good marks in the sciences and mathematics. A university education became available to Robert when his father was once again promoted and the family’s finances improved. Robert decided to study natural sciences at Göttingen University, close to his home. After two semesters, Koch transferred his field of study to medicine. He had dreams of becoming a physician on a ship. His father had traveled widely in Europe and passed a desire for travel on to his son. Although bacteriology was not taught then at the University, Koch would later credit his inter- est in that field to Jacob Henle, an anatomist who had pub- lished a theory of contagion in 1840. Many ideas about contagious diseases, particularly those of chemist and micro- biologist Louis Pasteur, who was challenging the prevailing myth of spontaneous generation, were still being debated in universities in the 1860s. During Koch’s fifth semester at medical school, Henle recruited him to participate in a research project on the struc- ture of uterine nerves. The resulting essay won first prize. It was dedicated to his father and bore the Latin motto, Nunquam Otiosus,, meaning never idle. During his sixth semester, he assisted Georg Meissner at the Physiological Institute. There he studied the secretion of succinic acid in animals fed only on fat. Koch decided to experiment on himself, eating a half- pound of butter each day. After five days, however, he was so sick that he limited his study to animals. The findings of this study eventually became Koch’s dissertation. In January 1866, he finished the final exams for medical school and graduated with highest distinction. After finishing medical school, Koch held various posi- tions; he worked as an assistant at a hospital in Hamburg, where he became familiar with cholera, and also as an assis- tant at a hospital for developmentally delayed children. In addition, he made several attempts to establish a private prac- tice. In July, 1867, he married Emmy Adolfine Josephine Fraatz, a daughter of an official in his hometown. Their only child, a daughter, was born in 1868. Koch finally succeeded in establishing a practice in the small town of Rakwitz where he settled with his family. Shortly after moving to Rakwitz, the Franco-Prussian War broke out and Koch volunteered as a field hospital physi- cian. In 1871, the citizens of Rakwitz petitioned Koch to return to their town. He responded, leaving the army to resume his practice, but he didn’t stay long. He soon took the exams to qualify for district medical officer and in August 1872 was appointed to a vacant position at Wollstein, a small town near the Polish border. It was here that Koch’s ambitions were finally able to flourish. Though he continued to see patients, Koch converted part of his office into a laboratory. He obtained a microscope and observed, at close range, the diseases his patients con- fronted him with. One such disease was anthrax, which is spread from animals to humans through contaminated wool, by eating uncooked meat, or by breathing in airborne spores emanating from contaminated products. Koch examined under the microscope the blood of infected sheep and saw specific microorganisms that confirmed a thesis put forth ten years earlier by biologist C. J. Davaine that anthrax was caused by a bacillus. Koch attempted to culture (grow) these bacilli in cattle blood so he could observe their life cycle, including their formation into spores and their germination. Koch per- formed scrupulous research both in the laboratory and in ani- mals before showing his work to Ferdinand Cohn, a botanist at the University of Breslau. Cohn was impressed with the work and replicated the findings in his own laboratory. He published Koch’s paper in 1876. In 1877, Koch published another paper that elucidated the techniques he had used to isolate Bacillus anthracis. He had dry-fixed bacterial cultures onto glass slides, then stained the cultures with dyes to better observe them, and pho- tographed them through the microscope. It was only a matter of time that Koch’s research eclipsed his practice. In 1880, he accepted an appointment as a government advisor with the Imperial Department of Health in Berlin. His task was to develop methods of isolating and womi_K 5/6/03 3:29 PM Page 326 Koch, Robert WORLD OF MICROBIOLOGY AND IMMUNOLOGY 327 • • cultivating disease-producing bacteria and to formulate strate- gies for preventing their spread. In 1881 he published a report advocating the importance of pure cultures in isolating dis- ease-causing organisms and describing in detail how to obtain them. The methods and theory espoused in this paper are still considered fundamental to the field of modern bacteriology. Four basic criteria, now known as Koch’s postulates, are essential for an organism to be identified as pathogenic, or capable of causing disease. First, the organism must be found in the tissues of animals with the disease and not in disease- free animals. Second, the organism must be isolated from the diseased animal and grown in a pure culture outside the body, or in vitro. Third, the cultured organism must be able to be transferred to a healthy animal, which will subsequently show signs of infection. And fourth, the organisms must be able to be isolated from the infected animal. While in Berlin, Koch became interested in tuberculosis, which he was convinced was infectious, and, therefore, caused by a bacterium. Several scientists had made similar claims but none had been verified. Many other scientists persisted in believing that tuberculosis was an inherited disease. In six months, Koch succeeded in isolating a bacillus from tissues of humans and animals infected with tuberculosis. In 1882, he published a paper declaring that this bacillus met his four con- ditions—that is, it was isolated from diseased animals, it was grown in a pure culture, it was transferred to a healthy animal who then developed the disease, and it was isolated from the animal infected by the cultured organism. When he presented his findings before the Physiological Society in Berlin on March 24, he held the audience spellbound, so logical and thor- ough was his delivery of this important finding. This day has come to be known as the day modern bacteriology was born. In 1883, Koch’s work on tuberculosis was interrupted by the Hygiene Exhibition in Berlin, which, as part of his duties with the health department, he helped organize. Later that year, he finally realized his dreams of travel when he was invited to head a delegation to Egypt where an outbreak of cholera had occurred. Louis Pasteur had hypothesized that cholera was caused by a microorganism; within three weeks, Koch had identified a comma-shaped organism in the intes- tines of people who had died of cholera. However, when test- ing this organism against his four postulates, he found that the disease did not spread when injected into other animals. Undeterred, Koch proceeded to India where cholera was also a growing problem. There, he succeeded in finding the same organism in the intestines of the victims of cholera, and although he was still unable to induce the disease in experi- mental animals, he did identify the bacillus when he exam- ined, under the microscope, water from the ponds used for drinking water. He remained convinced that this bacillus was the cause of cholera and that the key to prevention lay in improving hygiene and sanitation. Koch returned to Germany and from 1885–1890 was administrator and professor at Berlin University. He was highly praised for his work, though some high-ranking scien- tists and doctors continued to disagree with his conclusions. Koch was an adept researcher, able to support each claim with his exacting methodology. In 1890, however, Koch faltered from his usual perfectionism and announced at the International Medical Congress in Berlin that he had found an inoculum that could prevent tuberculosis. He called this agent tuberculin. People flocked to Berlin in hopes of a cure and Koch was persuaded to keep the exact formulation of tuber- culin a secret, in order to discourage imitations. Although opti- mistic reports had come out of the clinical trials Koch had set up, it soon became clear from autopsies that tuberculin was causing severe inflammation in many patients. In January 1891, under pressure from other scientists, Koch finally pub- lished the nature of the substance, but it was an uncharacteris- tically vague and misleading report which came under immediate criticism from his peers. Koch left Berlin for a time after this incident to recover from the professional setback, although the German govern- ment continued to support him throughout this time. An Institute for Infectious Diseases was established and Koch was named director. With a team of researchers, he continued his work with tuberculin, attempting to determine the ideal dose at which the agent could be the safest and most effective. The Robert Koch, whose postulates on the identification of microorganisms as the cause of a disease remain a fundamental underpinning of infectious microbiology. womi_K 5/6/03 3:29 PM Page 327 Koch’s postulates WORLD OF MICROBIOLOGY AND IMMUNOLOGY 328 • • discovery that tuberculin was a valuable diagnostic tool (caus- ing a reaction in those infected but none in those not infected), rather than a cure, helped restore Koch’s reputation. In 1892, there was a cholera outbreak in Hamburg. Thousands of peo- ple died. Koch advocated strict sanitary conditions and isola- tion of those found to be infected with the bacillus. Germany’s senior hygienist, Max von Pettenkofer, was unconvinced that the bacillus alone could cause cholera. He doubted Koch’s ideas, going so far as to drink a freshly isolated culture. Several of his colleagues joined him in this demonstration. Two developed symptoms of cholera, Pettenkofer suffered from diarrhea, but no one died; Pettenkofer felt vindicated in his opposition to Koch. Nevertheless, Koch focused much of his energy on testing the water supply of Hamburg and Berlin and perfecting techniques for filtering drinking water to pre- vent the spread of the bacillus. In the following years, he gave the directorship of the Institute over to one of his students so he could travel again. He went to India, New Guinea, Africa, and Italy, where he studied diseases such as the plague, malaria, rabies, and vari- ous unexplained fevers. In 1905, after returning to Berlin from Africa, he was awarded the Nobel Prize for physiology and medicine for his work on tuberculosis. Subsequently, many other honors were awarded him recognizing not only his work on tuberculosis, but his more recent research on tropical dis- eases, including the Prussian Order Pour le Merits in 1906 and the Robert Koch medal in 1908. The Robert Koch Medal was established to honor the greatest living physicians, and the Robert Koch Foundation, established with generous grants from the German government and from the American philan- thropist, Andrew Carnegie, was founded to work toward the eradication of tuberculosis. Meanwhile, Koch settled back into the Institute where he supervised clinical trials and production of new tuberculins. He attempted to answer, once and for all, the question of whether tuberculosis in cattle was the same disease as it was in humans. Between 1882 and 1901 he had changed his mind on this question, coming to accept that bovine tuberculosis was not a danger to humans, as he had previously thought. He presented his arguments at conferences in the United States and Britain during a time when many governments were attempting large-scale efforts to minimize the transmission of tuberculosis through limiting meat and milk. Koch did not live to see this question answered. On April 9, 1910, three days after lecturing on tuberculosis at the Berlin Academy of Sciences, he suffered a heart attack from which he never fully recovered. He died at Baden Baden the next month at the age of 67. He was honored after death by the naming of the Institute after him. In the first paper he wrote on tuberculo- sis, he stated his lifelong goal, which he clearly achieved: “I have undertaken my investigations in the interests of public health and I hope the greatest benefits will accrue therefrom.” See also Bacteria and bacterial infection; History of microbi- ology; History of public health; Koch’s postulates; Laboratory techniques in microbiology K OCH’S POSTULATES Koch’s postulates Koch’s postulates are a series of conditions that must be met for a microorganism to be considered the cause of a disease. German microbiologist Robert Koch (1843–1910) proposed the postulates in 1890. Koch originally proposed the postulates in reference to bacterial diseases. However, with some qualifications, the postulates can be applied to diseases caused by viruses and other infectious agents as well. According to the original postulates, there are four con- ditions that must be met for an organism to be the cause of a disease. Firstly, the organism must be present in every case of the disease. If not, the organism is a secondary cause of the infection, or is coincidentally present while having no active role in the infection. Secondly, the organism must be able to be isolated from the host and grown in the artificial and con- trolled conditions of the laboratory. Being able to obtain the microbe in a pure form is necessary for the third postulate that stipulates that the disease must be reproduced when the iso- lated organism is introduced into another, healthy host. The fourth postulate stipulates that the same organism must be able to be recovered and purified from the host that was experi- mentally infected. Since the proposal and general acceptance of the postu- lates, they have proven to have a number of limitations. For example, infections organisms such as some the bacterium Mycobacterium leprae, some viruses, and prions cannot be grown in artificial laboratory media. Additionally, the postu- lates are fulfilled for a human disease-causing microorganism by using test animals. While a microorganism can be isolated from a human, the subsequent use of the organism to infect a healthy person is unethical. Fulfillment of Koch’s postulates requires the use of an animal that mimics the human infection as closely as is possible. Another limitation of Koch’s postulates concerns instances where a microorganism that is normally part of the normal flora of a host becomes capable of causing disease when introduced into a different environment in the host (e.g., Staphylococcus aureus), or when the host’s immune system is malfunctioning (e.g., Serratia marcescens. Despite these limitations, Koch’s postulates have been very useful in clarifying the relationship between microorgan- isms and disease. See also Animal models of infection; Bacteria and bacterial infection; Germ theory of disease; History of immunology; History of microbiology; History of public health; Laboratory techniques in immunology; Laboratory tech- niques in microbiology KÖHLER, GEORGES (1946-1995) Köhler, Georges German immunologist For decades, antibodies, substances manufactured by the plasma cells to help fight disease, were produced artificially by injecting animals with foreign macromolecules, then womi_K 5/6/03 3:29 PM Page 328 Krebs, Hans Adolf WORLD OF MICROBIOLOGY AND IMMUNOLOGY 329 • • extracted by bleeding the animals and separating the anti- serum in their blood. The technique was arduous and far from foolproof. But the discovery of the hybridoma technique by German immunologist Georges Köhler changed revolutionize the procedure. Köhler’s work made antibodies relatively easy to produce and dramatically facilitated research on many seri- ous medical disorders such as acquired immunodeficiency syndrome (AIDS) and cancer. For his work on what would come to be known as monoclonal antibodies, Köhler shared the 1984 Nobel Prize in medicine. Born in Munich, in what was then occupied Germany, Georges Jean Franz Köhler attended the University of Freiburg, where he obtained his Ph.D. in biology in 1974. From there he set off to Cambridge University in England, to work as a postdoctoral fellow for two years at the British Medical Research Council’s laboratories. At Cambridge, Köhler worked under Dr. César Milstein, an Argentinean-born researcher with whom Köhler would eventually share the Nobel Prize. At the time, Milstein, who was Köhler’s senior by nineteen years, was a distinguished immunologist, and he actively encouraged Köhler in his research interests. Eventually, it was while working in the Cambridge laboratory that Köhler discovered the hybridoma technique. Dubbed by the New York Times as the “guided missiles of biology,” antibodies are produced by human plasma cells in response to any threatening and harmful bacterium, virus, or tumor cell. The body forms a specific antibody against each antigen; and César Milstein once told the New York Times that the potential number of different antigens may reach “well over a million.” Therefore, for researchers working to combat diseases like cancer, an understanding of how antibodies could be harnessed for a possible cure is of great interest. And although scientists knew the benefits of producing antibodies, until Köhler and Milstein published their findings, there was no known technique for maintaining the long-term culture of antibody-forming plasma cells. Köhler’s interest in the subject had been aroused years earlier, when he had become intrigued by the work of Dr. Michael Potterof the National Cancer Institute in Bethesda, Maryland. In 1962 Potter had induced myelomas, or plasma- cell tumors in mice, and others had discovered how to keep those tumors growing indefinitely in culture. Potter showed that plasma tumor cells were both seemingly immortal and able to create an unlimited number of identical antibodies. The only drawback was that there seemed no way to make the cells produce a certain type of antibody. Because of this, Köhler wanted to initiate a cloning experiment that would fuse plasma cells able to produce the desired antibodies with the “immor- tal” myeloma cells. With Milstein’s blessing, Köhler began his experiment. “For seven weeks after he had made the hybrid cells,” the New York Times reported in October, 1984, “Dr. Köhler refrained from testing the outcome of the experiment for fear of likely disappointment. At last, around Christmas 1974, he persuaded his wife,” Claudia Köhler, “to come to the win- dowless basement where he worked to share his anticipated disappointment after the critical test.” But disappointment turned to joy when Köhler discovered his test had been a suc- cess: Astoundingly, his hybrid cells were making pure anti- bodies against the test antigen. The result was dubbed mono- clonal antibodies. For his contribution to medical science, Köhler—who in 1977 had relocated to Switzerland to do research at the Basel Institute for Immunology—was awarded the Nobel in 1984. The implications of Köhler’s discovery were immense, and opened new avenues of basic research. In the early 1980s Köhler’s discovery led scientists to identify various lympho- cytes, or white blood cells. Among the kinds discovered were the T-4 lymphocytes, the cells destroyed by AIDS. Monoclonal antibodies have also improved tests for hepatitis B and streptococcal infections by providing guidance in selecting appropriate antibiotics, and they have aided in the research on thyroid disorders, lupus, rheumatoid arthritis, and inherited brain disorders. More significantly, Köhler’s work has led to advances in research that can harness monoclonal antibodies into certain drugs and toxins that fight cancer, but would cause damage in their own right. Researchers are also using monoclonal antibodies to identify antigens specific to the surface of cancer cells so as to develop tests to detect the spread of cancerous cells in the body. Despite the significance of the discovery, which has also resulted in vast amounts of research funds for many research laboratories, for Köhler and Milstein—who never patented their discovery—there was little financial remunera- tion. Following the award, however, he and Milstein, together with Michael Potter, were named winners of the Lasker Medical Research Award. In 1985, Köhler moved back to his hometown of Freiburg, Germany, to assume the directorship of the Max Planck Institute for Immune Biology. He died in Freiburg in 1995. See also Antibody-antigen, biochemical and molecular reac- tions; Antibody and antigen; Antibody formation and kinetics; Antibody, monoclonal; Immunity, active, passive and delayed; Immunity, cell mediated; Immunity, humoral regulation; Immunodeficiency; Immunodeficiency disease syndromes; Immunodeficiency diseases K REBS , HANS A DOLF (1900-1981) Krebs, Hans Adolf German biochemist Few students complete an introductory biology course without learning about the Krebs cycle, an indispensable step in the process the body performs to convert food into energy. Also known as the citric acid cycle or tricarboxylic acid cycle, the Krebs cycle derives its name from one of the most influential biochemists of our time. Born in the same year as the twenti- eth century, Hans Adolf Krebs spent the greater part of his eighty-one years engaged in research on intermediary metab- olism. First rising to scientific prominence for his work on the ornithine cycle of urea synthesis, Krebs shared the Nobel Prize for physiology and medicine in 1953 for his discovery of the citric acid cycle. Over the course of his career, the German- born scientist published, oversaw, or supervised a total of womi_K 5/6/03 3:29 PM Page 329 Krebs, Hans Adolf WORLD OF MICROBIOLOGY AND IMMUNOLOGY 330 • • more than 350 scientific publications. But the story of Krebs’s life is more than a tally of scientific achievements; his biogra- phy can be seen as emblematic of biochemistry’s path to recognition as its own discipline. In 1900, Alma Davidson Krebs gave birth to her second child, a boy named Hans Adolf. The Krebs family—Hans, his parents, sister Elisabeth and brother Wolfgang—lived in Hildesheim, in Hanover, Germany. There his father Georg practiced medicine, specializing in surgery and diseases of the ear, nose, and throat. Hans developed a reputation as a loner at an early age. He enjoyed swimming, boating, and bicycling, but never excelled at athletic competitions. He also studied piano diligently, remaining close to his teacher throughout his university years. At the age of fifteen, the young Krebs decided he wanted to follow in his father’s footsteps and become a physician. World War I had broken out, however, and before he could begin his medical studies, he was drafted into the army upon turning eighteen in August of 1918. The following month he reported for service in a signal corps reg- iment in Hanover. He expected to serve for at least a year, but shortly after he started basic training, the war ended. Krebs received a discharge from the army to commence his studies as soon as possible. Krebs chose the University of Göttingen, located near his parents’ home. There, he enrolled in the basic science cur- riculum necessary for a student planning a medical career and studied anatomy, histology, embryology and botanical science. After a year at Göttingen, Krebs transferred to the University of Freiburg. At Freiburg, Krebs encountered two faculty mem- bers who enticed him further into the world of academic research: Franz Knoop, who lectured on physiological chem- istry, and Wilhelm von Möllendorff, who worked on histolog- ical staining. Möllendorff gave Krebs his first research project, a comparative study of the staining effects of different dyes on muscle tissues. Impressed with Krebs’s insight that the effi- cacy of the different dyes stemmed from how dispersed and dense they were rather than from their chemical properties, Möllendorff helped Krebs write and publish his first scientific paper. In 1921, Krebs switched universities again, transferring to the University of Munich, where he started clinical work under the tutelage of two renowned surgeons. In 1923, he completed his medical examinations with an overall mark of “very good,” the best score possible. Inspired by his university studies, Krebs decided against joining his father’s practice as he had once planned; instead, he planned to balance a clinical career in medicine with experimental work. But before he could turn his attention to research, he had one more hurdle to complete, a required clinical year, which he served at the Third Medical Clinic of the University of Berlin. Krebs spent his free time at the Third Medical Clinic engaged in scientific investigations connected to his clinical duties. At the hospital, Krebs met Annelise Wittgenstein, a more experienced clinician. The two began investigating physical and chemical factors that played substantial roles in the distribution of substances between blood, tissue, and cere- brospinal fluid, research that they hoped might shed some light on how pharmaceuticals such as those used in the treat- ment of syphilis penetrate the nervous system. Although Krebs published three articles on this work, later in life he belittled these early, independent efforts. His year in Berlin convinced Krebs that better knowledge of research chemistry was essential to medical practice. Accordingly, the twenty-five-year-old Krebs enrolled in a course offered by Berlin’s Charité Hospital for doctors who wanted additional training in laboratory chemistry. One year later, through a mutual acquaintance, he was offered a paid research assistantship by Otto Warburg, one of the leading bio- chemists of the time. Although many others who worked with Warburg called him autocratic, under his tutelage Krebs devel- oped many habits that would stand him in good stead as his own research progressed. Six days a week work began at Warburg’s laboratory at eight in the morning and concluded at six in the evening, with only a brief break for lunch. Warburg worked as hard as the students. Describing his mentor in his autobiography, Hans Krebs: Reminiscences and Reflections, Krebs noted that Warburg worked in his laboratory until eight days before he died from a pulmonary embolism. At the end of his career, Krebs wrote a biography of his teacher, the sub- title of which described his perception of Warburg: “cell phys- iologist, biochemist, and eccentric.” Krebs’s first job in Warburg’s laboratory entailed famil- iarizing himself with the tissue slice and manometric (pressure measurement) techniques the older scientist had developed. Until that time, biochemists had attempted to track chemical processes in whole organs, invariably experiencing difficulties controlling experimental conditions. Warburg’s new tech- nique, affording greater control, employed single layers of tis- sue suspended in solution and manometers (pressure gauges) to measure chemical reactions. In Warburg’s lab, the tissue slice/manometric method was primarily used to measure rates of respiration and glycolysis, processes by which an organism delivers oxygen to tissue and converts carbohydrates to energy. Just as he did with all his assistants, Warburg assigned Krebs a problem related to his own research—the role of heavy metals in the oxidation of sugar. Once Krebs completed that project, he began researching the metabolism of human cancer tissue, again at Warburg’s suggestion. While Warburg was jealous of his researchers’ laboratory time, he was not stingy with bylines; during Krebs’s four years in Warburg’s lab, he amassed sixteen published papers. Warburg had no room in his lab for a scientist interested in pursuing his own research. When Krebs proposed undertaking studies of inter- mediary metabolism that had little relevance for Warburg’s work, the supervisor suggested Krebs switch jobs. Unfortunately for Krebs, the year was 1930. Times were hard in Germany, and research opportunities were few. He accepted a mainly clinical position at the Altona Municipal Hospital, which supported him while he searched for a more research-oriented post. Within the year, he moved back to Freiburg, where he worked as an assistant to an expert on metabolic diseases with both clinical and research duties. In the well-equipped Freiburg laboratory, Krebs began to test whether the tissue slice technique and manometry he had mas- tered in Warburg’s lab could shed light on complex synthetic metabolic processes. Improving on the master’s methods, he began using saline solutions in which the concentrations of womi_K 5/6/03 3:29 PM Page 330 Krebs cycle WORLD OF MICROBIOLOGY AND IMMUNOLOGY 331 • • various ions matched their concentrations within the body, a technique which eventually was adopted in almost all bio- chemical, physiological, and pharmacological studies. Working with a medical student named Kurt Henseleit, Krebs systematically investigated which substances most influenced the rate at which urea—the main solid component of mammalian urine—forms in liver slices. Krebs noticed that the rate of urea synthesis increased dramatically in the pres- ence of ornithine, an amino acid present during urine produc- tion. Inverting the reaction, he speculated that the same ornithine produced in this synthesis underwent a cycle of con- version and synthesis, eventually to yield more ornithine and urea. Scientific recognition of his work followed almost immediately, and at the end of 1932—less than a year and a half after he began his research—Krebs found himself appointed as a Privatdozent at the University of Freiburg. He immediately embarked on the more ambitious project of iden- tifying the intermediate steps in the metabolic breakdown of carbohydrates and fatty acids. Krebs was not to enjoy his new position in Germany for long. In the spring of 1933, along with many other German sci- entists, he found himself dismissed from his job because of Nazi purging. Although Krebs had renounced the Jewish faith twelve years earlier at the urging of his patriotic father, who believed wholeheartedly in the assimilation of all German Jews, this legal declaration proved insufficiently strong for the Nazis. In June of 1933, he sailed for England to work in the biochemistry lab of Sir Frederick Gowland Hopkins of the Cambridge School of Biochemistry. Supported by a fellowship from the Rockefeller Foundation, Krebs resumed his research in the British laboratory. The following year, he augmented his research duties with the position of demonstrator in biochem- istry. Laboratory space in Cambridge was cramped, however, and in 1935 Krebs was lured to the post of lecturer in the University of Sheffield’s Department of Pharmacology by the prospect of more lab space, a semi-permanent appointment, and a salary almost double the one Cambridge was paying him. His Sheffield laboratory established, Krebs returned to a problem that had long preoccupied him: how the body pro- duced the essential amino acids that play such an important role in the metabolic process. By 1936, Krebs had begun to suspect that citric acid played an essential role in the oxidative metabolism by which the carbohydrate pyruvic acid is broken down so as to release energy. Together with his first Sheffield graduate student, William Arthur Johnson, Krebs observed a process akin to that in urea formation. The two researchers showed that even a small amount of citric acid could increase the oxygen absorption rate of living tissue. Because the amount of oxygen absorbed was greater than that needed to completely oxidize the citric acid, Krebs concluded that citric acid has a catalytic effect on the process of pyruvic acid con- version. He was also able to establish that the process is cycli- cal, citric acid being regenerated and replenished in a subsequent step. Although Krebs spent many more years refin- ing the understanding of intermediary metabolism, these early results provided the key to the chemistry that sustains life processes. In June of 1937, he sent a letter to Nature reporting these preliminary findings. Within a week, the editor notified him that his paper could not be published without a delay. Undaunted, Krebs revised and expanded the paper and sent it to the new Dutch journal Enzymologia, which he knew would rapidly publicize this significant finding. In 1938, Krebs married Margaret Fieldhouse, a teacher of domestic science in Sheffield. The couple eventually had three children. In the winter of 1939, the university named him lecturer in biochemistry and asked him to head their new department in the field. Married to an Englishwoman, Krebs became a naturalized English citizen in September, 1939, three days after World War II began. The war affected Krebs’s work minimally. He con- ducted experiments on vitamin deficiencies in conscientious objectors, while maintaining his own research on metabolic cycles. In 1944, the Medical Research Council asked him to head a new department of biological chemistry. Krebs refined his earlier discoveries throughout the war, particularly trying to determine how universal the Krebs cycle is among living organisms. He was ultimately able to establish that all organ- isms, even microorganisms, are sustained by the same chemi- cal processes. These findings later prompted Krebs to speculate on the role of the metabolic cycle in evolution. In 1953, Krebs received the Nobel Prize in physiology and medicine, which he shared with Fritz Lipmann, the dis- coverer of co-enzyme A. The following year, Oxford University offered him the Whitley professorship in biochem- istry and the chair of its substantial department in that field. Once Krebs had ascertained that he could transfer his meta- bolic research unit to Oxford, he consented to the appoint- ment. Throughout the next two decades, Krebs continued research into intermediary metabolism. He established how fatty acids are drawn into the metabolic cycle and studied the regulatory mechanism of intermediary metabolism. Research at the end of his life was focused on establishing that the meta- bolic cycle is the most efficient mechanism by which an organism can convert food to energy. When Krebs reached Oxford’s mandatory retirement age of sixty-seven, he refused to end his research and made arrangements to move his research team to a laboratory established for him at the Radcliffe Hospital. Krebs died at the age of eighty-one. See also Cell cycle and cell division; Cell membrane transport KREBS CYCLE Krebs cycle The Krebs cycle is a set of biochemical reactions that occur in the mitochondria. The Krebs cycle is the final common path- way for the oxidation of food molecules such as sugars and fatty acids. It is also the source of intermediates in biosynthetic pathways, providing carbon skeletons for the synthesis of amino acids, nucleotides, and other key molecules in the cell. The Krebs cycle is also known as the citric acid cycle, and the tricarboxylic acid cycle. The Krebs cycle is a cycle because, during its course, it regenerates one of its key reactants. To enter the Krebs cycle, a food molecule must first be broken into two- carbon fragments known as acetyl groups, which are then joined to the carrier molecule coenzyme A womi_K 5/6/03 3:29 PM Page 331 Krebs cycle WORLD OF MICROBIOLOGY AND IMMUNOLOGY 332 • • (the A stands for acetylation). Coenzyme A is composed of the RNA nucleotide adenine diphosphate, linked to a pan- tothenate, linked to a mercaptoethylamine unit, with a termi- nal S-H.Dehydration of this linkage with the OH of an acetate group produces acetyl CoA. This reaction is cat- alyzed by pyruvate dehydrogenase complex, a large multi- enzyme complex. The acetyl CoA linkage is weak, and it is easily and irre- versibly hydrolyzed when Acetyl CoA reacts with the four- carbon compound oxaloacetate. Oxaloacetate plus the acetyl group form the six-carbon citric acid, or citrate. (Citric acid contains three carboxylic acid groups, hence the alternate names for the Krebs cycle.) Following this initiating reaction, the citric acid under- goes a series of transformations. These result in the formation of three molecules of the high-energy hydrogen carrier NADH (nicotinamide adenine dinucleotide), 1 molecule of another hydrogen carrier FADH2 (flavin adenine dinucleotide), 1 mol- ecule of high-energy GTP (guanine triphosphate), and 2 mol- ecules of carbon dioxide, a waste product. The oxaloacetate is regenerated, and the cycle is ready to begin again. NADH and FADH2 are used in the final stages of cellular respiration to generate large amounts of ATP. As a central metabolic pathway in the cell, the rate of the Krebs cycle must be tightly controlled to prevent too much, or too little, formation of products. This regulation occurs through inhibition or activation of several of the enzymes involved. Most notably, the activity of pyruvate dehydrogenase is inhib- ited by its products, acetyl CoA and NADH, as well as by GTP. This enzyme can also be inhibited by enzymatic addition of a phosphate group, which occurs more readily when ATP levels are high. Each of these actions serves to slow down the Krebs cycle when energy levels are high in the cell. It is important to note that the Krebs cycle is also halted when the cell is low on oxygen, even though no oxygen is consumed in it. Oxygen is needed further along in cell respiration though, to regenerate NAD+ and FAD. Without these, the cycle cannot continue, and pyruvic acid is converted in the cytosol to lactic acid by the fer- mentation pathway. The Krebs cycle is also a source for precursors for biosynthesis of a number of cell molecules. For instance, the synthetic pathway for amino acids can begin with either oxaloacetate or alpha-ketoglutarate, while the production of porphyrins, used in hemoglobin and other proteins, begins with succinyl CoA. Molecules withdrawn from the cycle for biosynthesis must be replenished. Oxaloacetate, for instance, can be formed from pyruvate, carbon dioxide, and water, with the use of one ATP molecule. See also Mitochondria and cellular energy womi_K 5/6/03 3:29 PM Page 332 L 333 • • LABORATORY TECHNIQUES IN IMMUNOLOGY Laboratory techniques in immunology Various laboratory techniques exist that rely on the use of antibodies to visualize components of microorganisms or other cell types and to distinguish one cell or organism type from another. Electrophoresis is a technique whereby the protein or carbohydrate components of microorganisms can be separated based upon their migration through a gel support under the driving influence of electricity. Depending upon the composi- tion of the gel, separation can be based on the net charge of the components or on their size. Once the components are sepa- rated, they can be distinguished immunologically. This appli- cation is termed immunoelectrophoresis. Immunoelectrophoresis relies upon the exposure of the separated components in the gel to a solution that contains an antibody that has been produced to one of the separated pro- teins. Typically, the antibody is generated by the injection of the purified protein into an animal such as a rabbit. For exam- ple, the protein that comprises the flagellar appendage of a certain bacteria can be purified and injected into the rabbit, so as to produce rabbit anti-flagellar protein. Immunoelectrophoresis can be used in a clinical immunology laboratory in order to diagnose illness, especially those that alter the immunoglobulin composition of body fluids. Research immunology laboratories also employ immu- noelectrophoresis to analyze the components of organisms, including microorganisms. One example of an immunoelectrophoretic technique used with microorganisms is known as the Western Blot. Proteins that have been separated on a certain type of gel sup- port can be electrically transferred to a special membrane. Application of the antibody will produce binding between the antibody and the corresponding antigen. Then, an antibody generated to the primary antibody (for example, goat anti-rab- bit antibody) is added. The secondary antibody will bind to the primary antibody. Finally, the secondary antibody can be con- structed so that a probe binds to the antibody’s free end. A chemical reaction produces a color change in the probe. Thus, bound primary antibody is visualized by the development of a dark band on the support membrane containing the elec- trophoretically separated proteins. Various controls can be invoked to ensure that this reaction is real and not the result of an experimental anomaly. A similar reaction can be used to detect antigen in sec- tions of biological material. This application is known as immunohistochemistry. The sections can be examined using either an electron microscope or a light microscope. The preparation techniques differ for the two applications, but both are similar in that they ensure that the antigen is free to bind the added antibody. Preservation of the antigen binding capacity is a delicate operation, and one that requires a skilled technician. The binding is visualized as a color reaction under light microscopic illumination or as an increased electron dense area under the electron beam of the electron micro- scope. The binding between antigen and antibody can be enhanced in light microscopic immunohistochemistry by the exposure of the specimen to heat. Typically a microwave is used. The heat energy changes the configuration of the antigen slightly, to ease the fit of the antigen with the antibody. However, the shape change must not be too great or the anti- body will not recognize the altered antigen molecule. Another well-establish laboratory immunological technique is known as enzyme-linked immunosorbent assay. The technique is typically shortened to ELISA. In the ELISA technique, antigen is added to a solid support. Antibody is flooded over the support. Where an antibody recognizes a corresponding antigen, binding of the two will occur. Next an antibody raised against the primary antibody is applied, and binding of the secondary antibody to the primary mole- cule occurs. Finally, a substrate is bound to a free portion of the secondary antibody, and the binding can be subsequently visualized as a color reaction. Typically, the ELISA test is womi_L 5/6/03 3:31 PM Page 333 Laboratory techniques in immunology WORLD OF MICROBIOLOGY AND IMMUNOLOGY 334 • • done using a plastic plate containing many small wells. This allows up to 100 samples to be tested in a single experiment. ELISA can reveal the presence of antigen in fluids such as a patient’s serum, for example. The nature of the antibody can be important in labora- tory immunological techniques. Antibodies such as those raised in a rabbit or a goat are described as being polyclonal in nature. That is, they do recognize a certain antigenic region. But if that region is present on different molecules, the antibody will react with all the molecules. The process of monoclonal antibody production can make antigenic identifi- cation much more specific, and has revolutionized immuno- logical analysis. Monoclonal antibodies are targeted against a single antigenic site. Furthermore, large amounts of the antibody can be made. This is achieved by fusing the antibody-producing cell obtained from an immunized mouse with a tumor cell. The resulting hybrid is known as a hybridoma. A particular hybridoma will mass-produce the antibody and will express the antibody on the surface of the cell. Because hybridoma cells are immortal, they grow and divide indefinitely. Hence the production of antibody can be ceaseless. Monoclonal antibodies are very useful in a clinical immunology laboratory, as an aid to diagnose diseases and to detect the presence of foreign or abnormal components in the blood. In the research immunology laboratory, monoclonal technology enables the specific detection of an antigenic tar- get and makes possible the development of highly specific vaccines. One example of the utility of monoclonal antibodies in an immunology laboratory is their use in the technique of flow cytometry. This technique separates sample as individual sam- ple molecules pass by a detector. Sample can be treated with monoclonal antibody followed by a second treatment with an antibody to the monoclonal to which is attached a molecule that will fluoresce when exposed to a certain wavelength of light. When the labeled sample passes by the detector and is illuminated (typically by laser light of the pre-determined wavelength), the labeled sample molecules will fluoresce. These can be detected and will be shunted off to a special col- lection receptacle. Many sorts of analyses are possible using flow cytometry, from the distinguishing of one type of bacte- ria from another to the level of the genetic material compris- ing such samples. See also Antibody-antigen, biochemical and molecular reac- tions Titration burettes are used to carefully control the pH of solutions used in laboratory procedures. womi_L 5/6/03 3:31 PM Page 334 [...]... Streptoco streptococcal infections LANDSTEINER, KARL Landsteiner, Karl (18 68 -1 9 43) American immunologist Karl Landsteiner was one of the first scientists to s physical processes of immunity He is best known for tification and characterization of the human blood gr B, and O, but his contributions spanned many a immunology, bacteriology, and pathology over a prolif year career Landsteiner identified the agents... the interaction of antig antibodies, and studied allergic reactions in experime Karl Landsteiner, awarded the 19 30 Nobel Prize in Medicine or Physiology for his discovery of human blood groups In 19 08, Landsteiner took charge of the departm pathology at the Wilhelmina Hospital in Vienna His te the hospital lasted twelve years, until March of 19 20 this time, Landsteiner was at the height of his career... brought in for autopsy Landsteiner took a portion of th spinal column and injected it into the spinal canal of • 18 85, at the age of 17 , Landsteiner passed the entrance examination for medical school at the University of Vienna He graduated from medical school at the age of 23 and immediately began advanced studies in the field of organic chemistry, working in the research laboratory of his mentor, Ernst... 18 97 During this time Landsteiner published his first article on the subject of bacteriology and serology, the study of blood Landsteiner moved to Vienna’s Institute of Pathology in 18 97, where he was hired to perform autopsies He continued to study immunology and the mysteries of blood on his own time In 19 00, Landsteiner wrote a paper in which he described the agglutination of blood that occurs when... adopted as stand cedure for treating wounds and during surgery Med Lister’s antiseptic method, which proved to be effect ing the Franco-Prussian War (18 70 18 71) In 18 77 became Professor of Surgery at King’s College, Lond Lister received many honors and awards A d surgeon, he treated both inflicted and surgical wou experimented with various antiseptics, developed ab sutures, and introduced a method of draining... less than 10 0 living bacteria per milliliter) does not induce luminescence, whereas luminescence is induced at a high cell density (e.g., 10 10 to 10 11 living bacteria per milliliter) The effect of cell density is particularly evident in those luminescent bacteria that live in the ocean When living free produce a chemical called homoserine lactone At densities, the chemical exits a bacterium and drifts... more than 90% of all reported vector-borne illnesses It is a significant public health problem and continues to be diagnosed in increasing numbers More than 99,000 cases were reported between 19 82 and 19 96 When the numbers for 19 96 Lyme disease cases reported were tallied, there were 16 ,455 new cases, a record high following a drop in reported cases from 19 94 (13 ,043 cases) to 19 95 (11 ,700 cases) Controversy... synthesis of proteins that increased the efficiency and accuracy of RNA synthesis All these ideas suggest that RNA was the primary substance of life and the later participation of DNA and proteins were later refinements that increased the survival potential of an already self-replicating living system Such a primordial pond where all these reactions were evolving eventually generated compartmentalization... “new depth of scientific understanding about the nature of life.” He foresaw advancements in the treatment of cancer, organ transplants, and geriatric medicine as presenting a whole new set of ethical and social problems, such as the availability and allocation of expensive health-care resources Lederberg was also interested in the study of biochemical life outside of Earth and coined the term exobiology... discovered caused foot -and- mouth disease This was the first hint that viruses existed At that time, Loeffler was working at the University of Greifswald as head of the department of hygiene Loeffler moved his laboratory to the island of the Insel Riems in order to safely continue his research on the disease In 19 13, Loeffler’s research took a back seat to his new position as director of the Robert Koch . tradition of Antoni van Leeuwenhoek (16 32 17 23). Outside Delft, he also drew on the legacy of Louis Pasteur (18 22 18 95), Robert Koch (18 43 19 10) , and Sergei Nikolayevich Winogradsky (18 56 19 53). Born. bacteria WORLD OF MICROBIOLOGY AND IMMUNOLOGY 336 • • Various biochemical tests are utilized in a microbiology laboratory. The ability of a microbe to utilize a particular com- pound and the nature of. 3: 31 PM Page 3 41 Lederberg, Joshua WORLD OF MICROBIOLOGY AND IMMUNOLOGY 342 • • In 19 47, while at Yale, Lederberg received an offer from the University of Wisconsin to become an assistant professor of