Photosynthesis: a short history of some modern experimental approaches.

This paper presents a personal interpretation of a chapter of plant physiology beginning from the early 1930s to the early 1940s, when plant physiologists tried to find the missing link between the two (dark and light) phases of photosynthesis. As initially inferred by Richard Willst?tter and Arthur Stoll in the 1910s, and then stated by Robert Emerson and William Arnold in 1932, the most accredited theory proposed that carbon dioxide must combine with chlorophyll in the dark. Successive light flashes activated the complex chlorophyll-carbon dioxide with oxygen evolution, and carbon dioxide was reduced to formaldehyde and successively polymerised into hexose. Arthur Stool in 1932 and Cornelius v. Niel in 1935 gave the first stroke to this theory suggesting that carbon dioxide must be reduced and assimilated by means of a process of water oxidation. Robert Hill showed the existence of an indissoluble link between the light phase, water oxidation and possibly oxygen evolution. Two physicists, Sam Ruben and Martin Kamen proposed the assembly of photosynthesis into a unitary process occurring as a sequence of several steps in the first 1940s. By utilising for the first time radioactive carbon (11C), they elaborated a new theory according to which carbon dioxide reduction was a repeated "cyclic" mechanism. This "heretical" view abolished the old, but still considered, theory of formaldehyde. Hill, Ruben and Kamen were able to exploit at best the possibility offered by a very advanced technology, thus confirming once again that ideas stand upon the powerful legs of technology.     

Relations between Light Level, Sucrose Concentration, and Translocation of Carbon 11 in Zea mays Leaves

The mechanism of carbon transport in Zea mays leaves was investigated using carbon 11 which is a short lived (half-life 20.4 min) positronemitting isotope. The gamma radiation produced on annihilation allows in vivo or nondestructive measurement of the isotope and the short half-life allows many measurements of translocation to be made on the same leaf within the same day.     

From Nuclear Science to Bacterial Cytochromes: the Work of Martin D. Kamen

Structural Bases for Function in Cytochromes c. An Interpretation of Comparative X-ray and Biochemical Data (Salemme, F. R., Kraut, J., and Kamen, M. D. (1973) J. Biol. Chem. 248, 7701–7716) Martin David Kamen (1913–2002) was born in Toronto, Canada, but grew up in Chicago. He enrolled at the University of Chicago in 1930, intending to study English. However, the Great Depression took a toll on his family''s finances, and his father suggested he switch his major to chemistry in order to make a living after graduating. By his junior year, Kamen was hooked on chemistry. He graduated in 1933 with honors in physical chemistry, working with William Draper Harkins to determine ammonia gas emission spectra excited by electrodeless discharge. He remained in Harkins'' lab for graduate school, earning his doctorate in physical chemistry in 1936 for an article on neutron scattering (1) that was accepted as his dissertation.Open in a separate windowMartin D. KamenBecause economic conditions were still bleak, Kamen followed the suggestion of one of his mentors, David Gans, and applied for a research post with Nobel laureate Ernest O. Lawrence, who had developed the cyclotron at the radiation laboratory in Berkeley, California. Kamen used his savings to move to Berkeley and worked at the laboratory without pay for 6 months before Lawrence offered him a staff position as a chemist. In addition to troubleshooting the cyclotrons and preparing samples of radioisotopes, Kamen performed numerous photosynthetic studies with Samuel Ruben, using carbon-11. Because carbon-11 had a half-life of only 21 min, Lawrence assigned Kamen and Ruben the task of finding carbon-14. The pair succeeded by bombarding graphite in the cyclotron, producing carbon-14, which had a 5730-year half-life (2).Kamen and Ruben planned to use their discovery to create a company that would construct and operate several cyclotrons dedicated to carbon-14 production and expand on the laboratory''s radioisotope program. However, the war intervened, and all non-war-related research at Berkeley was halted. Kamen was assigned to head a program studying the separation of uranium isotopes for the Manhattan Project. But, unexpectedly in 1944, he was declared a security risk and dismissed from the lab. A few years later, Kamen was called before the House Un-American Activities Committee, being wrongly linked to an espionage ring working for the USSR. Subsequently, the State Department refused to issue Kamen a passport, and the Chicago Tribune named him as a suspected spy. During the next decade, he fought recurring rumors and accusations that he had leaked atomic bomb secrets. Eventually, he won a libel suit against the Chicago Tribune, and the State Department reinstated his passport.In 1945, Kamen moved to the Mallinckrodt Institute of Radiology at the Washington University School of Medicine where he supervised cyclotron production of radioisotopes for medical research. His own research interests gradually shifted away from nuclear physics and radiochemistry to biochemistry, and he began several collaborations involving the use of radioisotopic tracers in biological and biomedical research.Kamen also initiated a series of experiments using carbon-14 to study photosynthesis in bacteria. This resulted in a number of important discoveries, including hydrogen photoevolution (3) and nitrogen fixation (4) in Rhodospirillum rubrum. While working with the bacteria, Kamen and Leo Vernon discovered that R. rubrum contained a c-type cytochrome (5), which they later named “cytochrome c₂.”Twenty years after it was discovered, the structure of cytochrome c₂ was solved (6). By comparing this structure with the recently solved structure of eukaryotic mitochondrial cytochrome c (7), Kamen and his colleagues were able to deduce information about the structural, functional, and evolutionary relationships in the cytochromes c. This is the subject of the Journal of Biological Chemistry (JBC) Classic reprinted here.Despite the fact that both eukaryotic cytochrome c and cytochrome c₂ serve analogous functions in their respective physiological electron transport chains, i.e. they both transport electrons to the terminal and most oxidizing electron carrier of each system, Kamen was able to find several differences between the molecules. For example, he noted that cytochrome c₂ has a more positive electrochemical potential and does not exhibit the large oxidation state-dependent conformational change characteristic of mitochondrial cytochrome c. Open in a separate windowKamen continued to study other bacterial cytochromes, showing that at least 12 subgroups of the cytochromes c exist. This resulted in new perspectives on potential variations in structure and function of the heme group in relation to protein.In 1957 Kamen moved to Brandeis University to help establish the graduate department of biochemistry, and in 1961 he joined the University of California, San Diego chemistry department to help found their new campus. He remained there until 1975, when he became director of the Chemical-Biological Development Laboratory at the University of Southern California. Kamen continued to teach into his eighties, being one of six faculty members of the Oregon Institute of Science and Medicine.Kamen received numerous awards and honors for his contributions to science, including the American Chemical Society''s Award for Applications of Nuclear Chemistry (1963), the American Society of Plant Physiologists'' Charles F. Kettering Research Award (1968), the American Society of Biological Chemists'' Merck Award (1982), the John Scott Medal of the City of Philadelphia (1988), the World Cultural Council''s Einstein Award (1990), and the U.S. Department of Energy''s Enrico Fermi Award (1996). He was a member of the National Academy of Sciences, the American Academy of Arts and Sciences, and the American Philosophical Society. ¹     

Inhibition of trehalose breakdown increases new carbon partitioning into cellulosic biomass in Nicotiana tabacum

Validamycin A was used to inhibit in vivo trehalase activity in tobacco enabling the study of subsequent changes in new C partitioning into cellulosic biomass and lignin precursors. After 12-h exposure to treatment, plants were pulse labeled using radioactive ¹¹CO₂, and the partitioning of isotope was traced into [¹¹C]cellulose and [¹¹C]hemicellulose, as well as into [¹¹C]phenylalanine, the precursor for lignin. Over this time course of treatment, new carbon partitioning into hemicellulose and cellulose was increased, while new carbon partitioning into phenylalanine was decreased. This trend was accompanied by a decrease in phenylalanine ammonia-lyase activity. After 4 d of exposure to validamycin A, we also measured leaf protein content and key C and N metabolite pools. Extended treatment increased foliar cellulose and starch content, decreased sucrose, and total amino acid and nitrate content, and had no effect on total protein.     

Following the path of carbon in photosynthesis: a personal story

Chronological recognition of the intermediates and mechanisms involved in photosynthetic carbon dioxide fixation is delineated. Sam Ruben and Martin Kamen's development of application of radioactive carbon for the study of carbon dioxide fixation provided impetus and techniques for following the path of carbon in photosynthesis. Discovery The identity of the primary carboxylation enzyme and its identity with the major protein of photosynthetic tissues (`Fraction 1' protein of Sam Wildman) is reviewed. Memories are dimmed by sixty years of exciting discoveries exploration in newer fields [see Benson 2002 (Annu Rev Plant Biol 53: 1–25), for research and perspectives beyond the early Berkeley days]. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of Radioiodination on Hormone Binding Ability

Abstract

This paper presents the effects of ¹²⁵I labelling on human chorionic gonadotropin binding ability. The results obtained showed a marked difference in the half-life of the specific radioactivity of the radiolabelled hormone comparing the one obtained by “self-displacement” analysis and that calculated considering the half-life of the radioactive isotope (60 days for ¹²⁵I). The decrease in the hormone half-life (t1/2= 3.10 days) was demonstrated by binding experiments performed at various days after hormone labelling. The affinity constant (Ka) and the number of binding sites obtained after Scatchard analysis using rat testes as receptor source, remained constant when the “self-displacement” specific radioactivity was used while they varied significantly if the 60 days half-life was considered for the data processing. The importance of the labelled hormone degradation as a possible cause of the decrease in the binding ability is also described.     

Use of 11alpha-deuterium labeled cortisol as a tracer for assessing reduced 11beta-HSD2 activity in vivo following glycyrrhetinic acid ingestion in a human subject

This study describes an oral administration of 5 mg of [1,2,4,19-13C4,11alpha-2H]cortisol (cortisol-13C4,2H1) to a human subject performed on two separate occasions, one with cortisol-13C4,2H1 alone and the other with cortisol-13C4,2H1 plus 130 mg per day of glycyrrhetinic acid for 6 days. The stable isotope methodology employed allowed for the evaluation of the individual in vivo activities of the two isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), 11beta-HSD1 and 11beta-HSD2, and to demonstrate the sensitivity of changes in cortisol elimination half-life for detecting inhibition of 11beta-HSD2 activity induced with glycyrrhetinic acid. The kinetic analysis associated with the loss of 11alpha-2H during the conversion of cortisol-13C4,2H1 to cortisone-13C4 by 11beta-HSD2 clearly indicated reduced 11beta-HSD2 activity with glycyrrhetinic acid ingestion, as observed by an increase in the elimination half-life of cortisol-13C4,2H1. The elimination half-life of cortisol-13C4,2H1 provided sensitive in vivo measures of 11beta-HSD2 activity and was more sensitive for detecting changes in renal 11beta-HSD2 activity than the measurement of the urinary ratio of free cortisol and free cortisone (UFF/UFE). The 2H-labeling in the 11alpha-position of cortisol served as an appropriate tracer for assessing the reduced 11beta-HSD2 activity in vivo induced by glycyrrhetinic acid.     

Measuring carbon gains from fungal networks in understory plants from the tribe Pyroleae (Ericaceae): a field manipulation and stable isotope approach

Partial mycoheterotrophy, a newly discovered form of mixotrophy in plants, has been described in at least two major lineages of angiosperms, the orchids and ericaceous plants in the tribe Pyroleae. Partial mycoheterotrophy entails carbon gains both directly from photosynthesis and via symbiotic mycorrhizal fungi, but determining the degree of plant dependence on fungal carbon is challenging. The purpose of this study was to determine if two chlorophyllous species of Pyroleae, Chimaphila umbellata and Pyrola picta, were receiving carbon via mycorrhizal networks and, if so, if their proportional dependency on fungal carbon gains increased under reduced light conditions. This was accomplished by a field experiment that manipulated light and plants' access to mycorrhizal networks, and by using the stable carbon isotope composition (δ(13)C) of leaf soluble sugars as a marker for the level of mycoheterotrophy. Based on leaf soluble sugars δ(13)C values, we calculated a site-independent isotope enrichment factor as a measure of fungal contributions to plant C. We found that, under each treatment and over time, the two test species demonstrated different isotopic responses caused by their different intrinsic physiologies. Our data, along with previously published studies, suggest that Chimaphila umbellata is primarily an autotrophic understory plant, while Pyrola picta may be capable of partial mycoheterotrophy. However, in this study, a 50% decrease in light availability did not significantly change the relative dependency of P. picta on carbon gains via mycoheterotrophy.     

Nitrogen gas exchange in the human knee

Human decompression sickness is presumed to result from excess inert gas in the body when ambient pressure is reduced. Although the most common symptom is pain in the skeletal joints, no direct study of nitrogen exchange in this region has been undertaken. For this study, nitrogen tagged with radioactive 13N was prepared in a linear accelerator. Nine human subjects rebreathed this gas from a closed circuit for 30 min, then completed a 40- to 100-min washout period breathing room air. The isotope 13N was monitored continuously in the subject's knee during the entire period using positron detectors. After correction for isotope decay (half-life = 9.96 min), the concentration in most knees continued to rise for at least 30 min into the washout period. Various causes of this unexpected result are discussed, the most likely of which is an extensive redistribution of gas within avascular knee tissues.     

Photoassimilate partitioning in nodulated soybean I. 11C methodology

An established method using ¹¹C for the in vivo measurement of photoassimilate partitioning within intact plants was applied to the characterization of partitioning of photoassimilate to soybean nodules. The method describes partitioning in terms of the magnitude and stability of partitioning flows, i.e. sink 'activity' and 'priority', and the transit time of tracer to a given sink. Leaflet labelling with ¹¹CO2 was recommended over whole shoot labelling to allow information on transport properties of the shoot to be acquired. The assumptions inherent in the method, that labelled and unlabelled photoassimilate in passage within the stem to the root system were well mixed and that tracer flow is unidirectional between source and sink (nodule), were validated. Tracer was re-exported from root to shoot, but this re-export process did not invalidate the assumption of unidirectional flow because the transit time of the re-export process was long relative to the half-life of the isotope. The transit time of tracer between entry to, and respiration from, the root system was also long (>60 min) relative to the half-life of the isotope. However, a significant fraction of tracer entering the root system was respired (c. 10% within 200 min), mainly by nodules (37% of tracer entering a nodule cluster was respired with 200 min). Therefore root-respired tracer was trapped and attributed to the nodule in partitioning calculations. A case study is presented using the method to assess changes in partitioning to nodules following treatment of the root system with nitrate, highlighting the limitation to this method of ontogenetic changes in the pattern of export from the load leaflet.     

A non-radioactive method for measuring Cu uptake in HepG2 cells

At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes 64Cu or 67Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, 63Cu and 65Cu (63Cu/65Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using 65Cu. The change in the 63Cu/65Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural 63Cu/65Cu ratio in HepG2 cells was altered using long-term incubation with 63Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 +/- 9.46 pmol microg DNA(-1) (week 1) to 155 +/- 8.63 pmol microg DNA(-1) (week 2) and stabilising at 171 +/- 4.82 pmol microg DNA(-1) (week 3). After three weeks of culture with 2 microM 63Cu the 63Cu/65Cu changed from 2.18 +/- 0.05 to 15.3 +/- 1.01. Cu uptake was then investigated as before using 65Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells.     

Effects of elemental composition on the incorporation of dietary nitrogen and carbon isotopic signatures in an omnivorous songbird

The use of stable isotopes to infer diet requires quantifying the relationship between diet and tissues and, in particular, knowing of how quickly isotopes turnover in different tissues and how isotopic concentrations of different food components change (discriminate) when incorporated into consumer tissues. We used feeding trials with wild-caught yellow-rumped warblers (Dendroica coronata) to determine delta15N and delta13C turnover rates for blood, delta15N and delta13C diet-tissue discrimination factors, and diet-tissue relationships for blood and feathers. After 3 weeks on a common diet, 36 warblers were assigned to one of four diets differing in the relative proportion of fruit and insects. Plasma half-life estimates ranged from 0.4 to 0.7 days for delta13C and from 0.5 to 1.7 days for delta15N . Half-life did not differ among diets. Whole blood half-life for delta13C ranged from 3.9 to 6.1 days. Yellow-rumped warbler tissues were enriched relative to diet by 1.7-3.6% for nitrogen isotopes and by -1.2 to 4.3% for carbon isotopes, depending on tissue and diet. Consistent with previous studies, feathers were the most enriched and whole blood and plasma were the least enriched or, in the case of carbon, slightly depleted relative to diet. In general, tissues were more enriched relative to diet for birds on diets with high percentages of insects. For all tissues, carbon and nitrogen isotope discrimination factors increased with carbon and nitrogen concentrations of diets. The isotopic signature of plasma increased linearly with the sum of the isotopic signature of the diet and the discrimination factor. Because the isotopic signature of tissues depends on both elemental concentration and isotopic signature of the diet, attempts to reconstruct diet from stable isotope signatures require use of mixing models that incorporate elemental concentration.     

Carbon-use efficiency in green sinks is increased when a blend of apoplastic fructose and glucose is available for uptake

Understanding how green sink strength is regulated in planta poses a difficult problem because non-structural carbohydrate (NSC) levels can have integrated, simultaneous feedback effects on photosynthesis, sugar uptake, and respiration that depend on specific NSC moieties. Photosynthetic gametophytes of the fern Ceratopteris richardii provide a simple land plant model to assess how different NSCs imported from the apoplast of intact plants affect green sink strength. Sink strength was quantified as the amount of exogenous sugar that plants grown in low light depleted from their liquid media, and the relative contributions of carbon assimilation by photosynthesis and sugar uptake was estimated from stable isotope analysis of plant dry mass. Gametophytes absorbed fructose and glucose with equal affinity when cultured on either hexose alone, or in the presence of an equimolar blend of both sugars. Plants also depleted sucrose from the surrounding media, although a portion of this disaccharide that was hydrolysed into fructose and glucose by putative cell wall invertase activity remained in the media. The δ(13)C in plant dry masses harvested from sugar treatments were all close to -18‰, indicating that 25-39% of total plant carbon was from C3 photosynthesis (δ(13)C=-29‰) and 61-75% was from uptake of exogenous sugars (δ(13)C=-11‰). Carbon-use efficiency (i.e. carbon accumulated/carbon depleted) was significantly improved when plants had a blend of exogenous sugars available compared with plants grown in a single hexose alone. Plants avoided complete down-regulation of photosynthesis even though a large excess of exogenous carbon fluxed through their cells.     

13C and 15N isotope effects for conversion of L-dihydroorotate to N-carbamyl-L-aspartate using dihydroorotase from hamster and Bacillus caldolyticus

In the pyrimidine biosynthetic pathway, N-carbamyl-L-aspartate (CA-asp) is converted to L-dihydroorotate (DHO) by dihydroorotase (DHOase). The mechanism of this important reaction was probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and 45 degrees C for hamster DHOase; 37, 50, and 60 degrees C for Bacillus caldolyticus), and the product CA-asp was purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the beta-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary and secondary isotope effects for the reaction catalyzed by DHOase showed little variation between enzymes or temperatures, the primary 13C and 15N isotope effects being approximately 1% on average, while the secondary 13C isotope effect is negligible or very slightly normal (>1.0000). These data indicate that the chemistry is at least partially rate-limiting while the secondary isotope effects suggest that the transition state may have lost some bending and torsional modes leading to a slight lessening of bond stiffness at the carbonyl carbon of the amide of CA-asp. The equilibrium isotope effects for DHO --> CA-asp have also been measured (secondary 13K(eq) = 1.0028 +/- 0.0002, primary 13K(eq) = 1.0053 +/- 0.0003, primary 15K(eq) = 1.0027 +/- 0.0003). Using these equilibrium isotope effects, the kinetic isotope effects for the physiological reaction (CA-asp --> DHO) have been calculated. These values indicate that the carbon of the amide group is more stiffly bonded in DHO while the slightly lesser, but still normal, values of the primary kinetic isotope effect show that the chemistry remains at least partially rate-limiting for the physiological reaction. It appears that the ring opening and closing is the slow step of the reaction.     

Novel 13C enrichment technique reveals early turnover of DHA in peripheral tissues

The brain is rich in DHA, which plays important roles in regulating neuronal function. Recently, using compound-specific isotope analysis that takes advantage of natural differences in carbon-13 content (¹³C/¹²C ratio or δ¹³C) of the food supply, we determined the brain DHA half-life. However, because of methodological limitations, we were unable to capture DHA turnover rates in peripheral tissues. In the current study, we applied compound-specific isotope analysis via high-precision GC combustion isotope ratio mass spectrometry to determine half-lives of brain, liver, and plasma DHA in mice following a dietary switch experiment. To model DHA tissue turnover rates in peripheral tissues, we added earlier time points within the diet switch study and took advantage of natural variations in the δ¹³C-DHA of algal and fish DHA sources to maintain DHA pool sizes and used an enriched (uniformly labeled ¹³C) DHA treatment. Mice were fed a fish-DHA diet (control) for 3 months, then switched to an algal-DHA treatment diet, the ¹³C enriched-DHA treatment diet, or they stayed on the control diet for the remainder of the study time course. In mice fed the algal and ¹³C enriched-DHA diets, the brain DHA half-life was 47 and 46 days, the liver half-life was 5.6 and 7.2 days, and the plasma half-life was 4.7 and 6.4 days, respectively. By using improved methodologies, we calculated DHA turnover rates in the liver and plasma, and our study for the first time, by using an enriched DHA source (very high δ¹³C), validated its utility in diet switch studies.     

Use of positron-emitting tracer imaging system for measuring the effect of salinity on temporal and spatial distribution of C tracer and coupling between source and sink organs

Salinity stress affects photosynthate partitioning between sources and sinks of plants, but how it affects these systems is less well understood. Because sources and sinks are closely tied, any adverse effect under suboptimal conditions on one of these is often misinterpreted for an effect on the other. Carbon partitioning is indispensable for stress resistance and good plant growth. In the present study, carbon partitioning in tomato plants (Lycopersicon esculentum L. cv. Momotarou) in a saline (NaCl) environment was studied by feeding radioactive ¹¹C and stable ¹³C isotopes. Pulse-chases were conducted to measure the spatial and temporal distribution of ¹³C. ¹³C was measured by a standard conventional technique, but ¹¹C distribution was monitored using a positron-emitting tracer imaging system (PETIS). Salt stress resulted in reduced carbon translocation toward roots. The majority of the photosynthate accumulated in the leaf. We also observed that the reduction in translocation of carbon occurred well before the salt stress symptoms of reduced photosynthesis and reduced plant growth in salt-exposed plants. The effect on sink activity was also shown by a decrease in stem diameter. In addition, PETIS analysis of ¹¹C translocation indicated that carbon translocation to roots was inhibited under salt conditions without a direct effect on leaf Na accumulation or osmotic stress. These results suggest that NaCl has direct effects on plants, inhibiting carbon partitioning within a few hours of salt exposure without inhibition of source activity.     

Glucose isotope, carbon recycling, and gluconeogenesis using [U-13C]glucose and mass isotopomer analysis

Experimental determinations of glucose carbon recycling using 14C or 13C glucose tracer often underestimate true Cori cycle activity because of dilution and exchange of isotope tracer through the tricarboxylic acid (TCA) cycle. The term glucose isotope recycling therefore is used to distinguish recycling of isotope from recycling of glucose carbon, the actual quantity of circulating glucose recycled. Recently, per-labeled glucose ([U-13C6]glucose) has been used to estimate glucose appearance rate and glucose isotope recycling. Chemical structural information determined by mass isotopomer analysis has been used to correct for dilution of isotope through the TCA cycle. In this report, we present experiments in the study of glucose turnover and recycling using [U-13C6]glucose. Methods of single injection and continuous infusion of [U-13C6]glucose are compared. A formula for the calculation of a dilution factor using TCA cycle parameters is derived. In this study of six rabbits, glucose turnover rate ranged from 3.4 to 8.8 mg/kg/min, and glucose m + 3 mass isotopomer recycling from 7 to 12%. The rate of pyruvate carboxylation (Y) was comparable to that of citrate synthetase, having an average relative flux of 0.89. Applying the correction factor for tracer dilution to the observed mass isotopomer recycling, we determined glucose carbon recycling (or Cori cycle activity) to be 22-35% of hepatic glucose output.     

Changes in the half-life of ribosomal protein messenger RNA caused by translational repression

The half-life of ribosomal protein operon L11 mRNA in vivo was measured during exponential growth by following the kinetics of incorporation of radioactive precursors into L11 mRNA transcribed from multi-copy plasmids. The degree of translational feedback regulation by L1, the L11 operon-specific translational repressor protein, was changed by altering the site on the "L11 mRNA" where L1 interacts. The half-life of the overproduced L11 mRNA increased by about fivefold when translational repression was abolished, while the half-life of mRNA from the spc ribosomal protein operon, which is not translationally regulated by L1, stayed constant. Furthermore, the half-life of L11 operon mRNA carrying an additional mutation in the ribosome binding site that abolishes translation remains short. This indicates that the change in half-life observed during increased gene dosage is due to translational repression by L1 and is probably a consequence of L1 blocking translation of L11 mRNA and not due to some nucleolytic activity mediated by L1.     

A latex agglutination test for the detection of Mycoplasma pneumoniae in respiratory exudates: a comparative study with a commercially available DNA-probe test.

We prepared polyclonal antibody specific to Mycoplasma pneumoniae. Using this antibody, we developed a latex agglutination test (LAT) for detecting the organism in respiratory exudates as rapid diagnosis of M. pneumoniae infection. Further, LAT was compared with DNA-probe test (DP) which was the only commercially available test for the rapid detection of the organism. In LAT, both M. pneumoniae and M. genitalium give positive agglutination, but the titer of M. genitalium was significantly lower than that of M. pneumoniae. The detection limit of LAT was 2 x 10(5) CFU/ml and that of DP was 5 x 10(4) CFU/ml in vitro. It was considered that target molecules in LAT were accumulated in the pharyngeal portion of the patients, because of their long half-life at 37 C. However, ribosomal RNA which was target molecule in DP was destroyed at 37 C much sooner, and the accumulation could not be expected. Actually, positive rate in LAT was higher than that in DP among clinical specimens in which M. pneumoniae was detected by culture method. The procedure of LAT is much easier and more rapid than that of DP in which radioactive isotope is required. LAT could be the choice of test for rapid diagnosis of M. pneumoniae infection.     

Evidence of substantial carbon isotope fractionation among substrate, inorganic carbon, and biomass during aerobic mineralization of 1, 2-dichloroethane by Xanthobacter autotrophicus

Carbon isotope fractionation during aerobic mineralization of 1, 2-dichloroethane (1,2-DCA) by Xanthobacter autotrophicus GJ10 was investigated. A strong enrichment of (13)C in residual 1,2-DCA was observed, with a mean fractionation factor alpha +/- standard deviation of 0.968 +/- 0.0013 to 0.973 +/- 0.0015. In addition, a large carbon isotope fractionation between biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor alpha to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of (13)C in 1,2-DCA arises because the first irreversible step of the initial enzymatic transformation of 1,2-DCA consists of an S(N)2 nucleophilic substitution. S(N)2 reactions are accompanied by a large kinetic isotope effect. The substantial carbon isotope fractionation between biomass and inorganic carbon could be explained by the kinetic isotope effect associated with the initial 1,2-DCA transformation and by the metabolic pathway of 1,2-DCA degradation. Carbon isotope fractionation during 1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass with characteristic carbon isotope compositions, which may be used to trace the process in contaminated environments.