Pat Unkefer

Los Alamos National Laboratory | LANL · Bioscience Division

A metabolite of ammonium assimilation was previously theorized to be involved in the coordination of the overall nitrate response in plants. Here we show that 2-hydroxy-5-oxoproline, made by transamination of glutamine, the first product of ammonium assimilation, may be involved in signaling a plant's ammonium assimilation status. In leaves, 2-hydr...

In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium began, little was known about the molecular basis of algal biomass or oil production. Very few algal genome sequences were available and efforts to identify the best-producing wild species through bioprospecting approaches had largely stalled after the U.S....

The clustering of genes in a pathway and the co-location of functionally related genes is widely recognized in prokaryotes. We used these characteristics to predict the metabolic involvement for a Transcriptional Regulator (TR) of unknown function, identified and confirmed its biological activity. A software tool that identifies the genes encoded w...

The main objective of NAABB was to combine science, technology, and engineering expertise from across the nation to break down critical technical barriers to commercialization of algae-based biofuels. The approach was to address technology development across the entire value chain of algal biofuels production, from selection of strains to cultivati...

The present invention describes a composition including a mixture of L- and D-pyroglutamate stereoisomers in a ratio of L to D of from about 80:20 to about 97:3 , and, a carrier medium for application of the L- and D-pyroglutamate stereoisomers to a target plant. The composition can also be used as a germination medium and may be incorporated into...

The reactivity of given metabolites is assessed using selected empirical atomic properties in the potential reaction center. Metabolic reactions are represented as biotransformation rules. These rules are generalized from the patterns in reactions. These patterns are not unique to reactants but are widely distributed among metabolites. Using a meta...

Transcriptional regulators (TRs) are an important and versatile group of proteins, yet very little progress has been achieved towards the discovery and annotation of their biological functions. We have characterized a previously unknown organic hydroperoxide resistance regulator from Burkholderia xenovoransLB400, Bxe_B2842, which is homologous to E...

To study the transcriptional regulation of oxidative tryptophan degradation in Burkholderia, we used comparative genomics that focused on the operon containing the genes annotated as kynA, kynU and kynB. In all sequenced β-proteobacteria to-date, including Burkholderia, Ralstonia, Collimonas, and Cupriavidus species, there is a conserved AsnC/Lrp f...

The search for effectors of novel transcriptional regulators is a challenging task. Here, we present the prediction and validation of an effector for a novel transcriptional regulator (TR). The clustering of genes around the gene coding for Bxe_A0425, a TR in Burkholderia xenovorans LB400 and its closest orthologs, suggests the conservation of a fu...

We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector m...

Transcriptional Regulators (TR) regulate and control the physiological processes that dictate phenotypic traits. Regulation occurs in response to stimuli, this response manifests itself in the binding of a effector molecule by the TR. Function prediction and possible effector library formulation are key factors in the discovery of the biological fu...

To uncover the transcriptional regulation of oxidative tryptophan degradation, comparative genomics were used which focused on the operon containing the genes kynA, kynU and kynB . In β and γ‐proteobacteria, including Burkholderia, Ralstonia, Collimonas , Cupriavidus and Pseudomonas species, there is a conserved asnC/lrp family transcriptional regu...

Determining transcription factor (TF) recognition motifs or operator sites is central to understanding gene regulation, yet few operators have been characterized. In this study, we used a protein-binding microarray (PBM) to discover the DNA recognition sites and putative regulons for three TetR and one MarR family TFs derived from Burkholderia xeno...

Our knowledge of the metabolites in cells and their reactions is far from complete as revealed by metabolomic measurements that detect many more small molecules than are documented in metabolic databases. Here, we develop an approach for predicting the reactivity of small-molecule metabolites in enzyme-catalyzed reactions that combines expert knowl...

Bars indicate the frequencies of triplet sequences that appear in non-coding regions of the E. coli genome. As can be seen, the non-coding genome sequence is not random, i.e., the assumption that sequences appear with equal probability is invalid. (3.93 MB TIF)

Example showing different feature vectors given different nucleotide environments: G in TGG and G in AGA. Cross reference with Figs. 2 and 3. (0.23 MB DOC)

Computational results for 44 TFs documented in DPInteract. Here, the cutoff values for the BvH, Match, and MATRIX SEARCH methods were each set to the lowest scoring sequence in the training set from which a model for a TF binding site was built. This approach, which guarantees that positive examples used in training are correctly classified, is dif...

Area under the curve (AUC) analysis for transcription factors (TFs) in RegulonDB with at least 20 known binding sites. A receiver operating characteristics (ROC) curve is a two-dimensional plot of the false positive rate (1 - specificity) versus the true positive rate (sensitivity). The AUC for an ROC curve is between 0 and 1. A perfect classifier...

Probe types from GRID [38] used to estimate the molecular interaction field features Pi and Qi for probe type i as described in Fig. 1 and the Methods section. Definitions of the minimum interaction energy, Pi, and interaction score, Qi, are given in the Methods section (Eq. 2). (0.14 MB DOC)

Correlation of propeller feature from simulation and experimental DNA structure. We downloaded all asymmetric units of nucleic acid-containing structures determined by X-ray crystallography from the Nucleic Acid Database (http://ndbserver.rutgers.edu/) [33]. From these structures, we extracted 1,867 3D DNA structures. For each DNA structure, we use...

Computational results for 54 TFs, whose number of known binding sites documented in RegulonDB is five or more. For each computational method, the training set size and the number of predicted binding sites are given. See http://cellsignaling.lanl.gov/EcoliTFs/SiteSleuth/ for the complete listing of binding sites predicted by each method for each TF...

For each method, mean cross-validation score (V), which is defined as the fraction of positive examples predicted to be true binding sites, for 54 TFs whose number of known binding sites documented in RegulonDB is five or more. (0.26 MB DOC)

An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF). Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. H...

The biosynthesis of the 3,4-dihydroxybenzoate moieties of the siderophore petrobactin, produced by B. anthracis str. Sterne, was probed by isotopic feeding experiments in iron-deficient media with a mixture of unlabeled and D-[(13)C6]glucose at a ratio of 5:1 (w/w). After isolation of the labeled siderophore, analysis of the isotopomers was conduct...

Stable isotope labeling of small-molecule metabolites (e.g. (13)C-labeling of glucose) is a powerful tool for characterizing pathways and reaction fluxes in a metabolic network. Analysis of isotope labeling patterns requires knowledge of the fates of individual atoms and moieties in reactions, which can be difficult to collect in a useful form when...

We investigate the ability of algorithms developed for reverse engineering of transcriptional regulatory networks to reconstruct metabolic networks from high-throughput metabolite profiling data. For benchmarking purposes, we generate synthetic metabolic profiles based on a well-established model for red blood cell metabolism. A variety of data set...

Our knowledge of metabolism is far from complete, and the gaps in our knowledge are being revealed by metabolomic detection of small-molecules not previously known to exist in cells. An important challenge is to determine the reactions in which these compounds participate, which can lead to the identification of gene products responsible for novel...

Soils are the major pool of terrestrial C globally. Estimating inventories and detecting changes in the soil C pool have remained elusive, largely because the spatial distribution of soil C varies considerably. New approaches are needed that enable more rapid, cost-effective, and sensitive measurements of soil C and that reduce uncertainty in estim...

Soil structural and water transmission properties, as influenced by land use and soil management, affect the coefficients of infiltration predictive models. Ten infiltration models were analyzed to assess these coefficients. The models tested include Green and Ampt (1911), Kostiakov (1932), Horton (1940), Mezencev (1948), Philip (1957), Holtan (196...

Effects of land use and management treatments on soil mechanical and hydrological properties were assessed by analysis of bulk and core soil samples and water infiltration measurements in the field, using double ring infiltrometers in five plots located at the experimental farm of the North Appalachian Experimental Watersheds (NAEW) near Coshocton,...

Increasing the concentration of prolines, such as 2-hydroxy-5-oxoproline, in the foliar portions of plants has been shown to cause an increase in carbon dioxide fixation, growth rate, dry weight, nutritional value (amino acids), nodulation and nitrogen fixation, photosynthetically derived chemical energy, and resistance to insect pests over the sam...

© 2002 Optical Society of America

Accurate measurement of carbon in soils remains one of the largest challenges in understanding sources and sinks of carbon in terrestrial systems. Conventional methods of analyzing carbon require large initial volumes of soil, therefore obtaining samples for conventional analysis is time consuming and expensive. The application of laser-induced bre...

Improving estimates of carbon inventories in soils is currently hindered by lack of a rapid analysis method for total soil carbon. A rapid, accurate, and precise method that could be used in the field would be a significant benefit to researchers investigating carbon cycling in soils and dynamics of soil carbon in global change processes. We tested...

The interactions between Pseudomonas syringae pv. tabaci and either nodulating alfalfa (Medicago sativa) or oat (Avena sativa) seedlings were examined to further our understanding of this rhizosphere association. P. syringae pv. tabaci produces and releases a toxin, tabtoxinine-β-lactam (TβL), that inactivates glutamine synthetase (GS). Sinorhizobi...

Rapid measurement of total carbon in soils is an important factor in modeling the effects of global change and carbon sequestration in soils. Conventional methods of carbon analysis such as dry combustion are relatively slow, and reliable estimation of carbon concentrations at the landscape scale is practically impossible because of the need for ma...

Photosynthesis and respiration are the largest fluxes into and out of the biosphere (Molles 1999). Consequently, small changes in these fluxes can potentially produce large changes in the storage of carbon in the biosphere. Terrestrial carbon fluxes account for more than half of the carbon transferred between the atmosphere and the earth's surface...

Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried ou...

Biotransformation of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) in slurry reactors was studied to determine the importance of supplementation of known biodegraders and the type of nutrient source required. Although addition of bacteria to the system increased the biotransformation rates, the increase may not justify the additional work and cost...

Asparagine, the primary assimilation product from N2 fixation in temperate legumes and the predominant nitrogen transport product in many plant species, is synthesized via asparagine synthetase (AS; EC 6.3.5.4). Here, we report the isolation and characterization of a cDNA and a gene encoding the nodule-enhanced form of AS from alfalfa. The AS gene...

The biotransformation of hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) has been observed in liquid culture by a consortium of bacteria found in horse manure. Five types of bacteria were found to predominate in the consortium and were isolated. The most effective of these isolates at transforming RDX was Serratia marcescens. The biotransformation of...

Low-level mixed radioactive wastes containing cellulose-based materials and plutonium have been generated during various nuclear processing activities. Biological digestion of the organic- or cellulose- based material was examined as an environmentally acceptable and effective method of treatment for these and other similar wastes. Cellulase enzyme...

Many microbes reduce the nitro substituents of 2,4,6-trinitrotoluene (TNT), producing aminodinitrotoluenes (ADNTs). These compounds are recalcitrant to further breakdown and are acutely toxic. In a search for organisms capable of metabolizing ADNTs, a bacterial strain was isolated for the ability to use 2-aminobenzoate (anthranilate) as sole C-sour...

Mycobacterium vaccae strain JOB-5 cometabolized 2,4,6-trinitrotoluene (TNT) in the presence of propane as a carbon and energy source. Two novel oxidized metabolites, as well as several known reduced products, were generated during catabolism of TNT by M. vaccae. During the cometabolic process, there was transient production of a brown chromophore....

Three species of the family Enterobacteriaceae that biochemically reduced hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) were isolated from nitramine explosive-contaminated soil. Two isolates, identified as Morganella morganii and Providencia rettgeri, completely transformed both RDX and the...

The DOE complex has generated vast quantities of complex heterogeneous mixed wastes. Paint stripper waste (PSW) is a complex waste that arose from decontamination and decommissioning activities. It contains paint stripper, cheesecloth, cellulose-based paints with Pb and Cr, and suspect Pu. Los Alamos National Laboratory has 150--200 barrels of PSW...

In dismantling weapons from stockpile reduction, environmentally acceptable methods to degrade the associated high explosive (BE) waste to non-energetic forms is a critical objective. Base hydrolysis appears to be a safe, simple, and inexpensive method for converting energetic materials (EN-MATs) into non-energetic materials. We have demonstrated t...

A glutamine synthetase (GS) cDNA isolated from an alfalfa cell culture cDNA library was found to represent a cytoplasmic GS. The full-length alfalfa GS1 coding sequence, in both sense and antisense orientation and under the transcriptional control of the cauliflower mosaic virus 35S promoter, was introduced into tobacco. Leaves of tobacco plants tr...

Ammonia assimilation has been implicated as participating in regulation of nitrogen fixation in free-living bacteria. In fact, these simple organisms utilize an integrated regulation of carbon and nitrogen metabolism; we except to observe an integration of nitrogen and carbon fixation in plants; how could these complex systems grow efficiently and...

The extensive manufacture, packing, and the use of explosives has often resulted in significant contamination of soils and ground waters near these activities. Congressional mandate has now required that such sites be remediated. An especially promising technology for this explosives problem is biotechnology. When applicable, biotechnology is cheap...

The analysis of stable sulfur and nitrogen isotopes can provide useful information in many types of environmental studies. For example, these elements are emitted in oxidized forms from air pollution sources and are deposited in appreciable quantities over vast regions of industrialized countries. Extensive research efforts in many countries are un...

Pseudomonas syringae pv. tabaci, a commonly recognized leaf pathogen of tobacco, can infest the rhizosphere of many plants, including oats. Normal oat plants do not survive this infestation as a consequence of the complete and irreversible inactivation of all of their glutamine synthetases by tabtoxinine-beta-lactam (TbetaL), a toxin released by pv...

Several lines of evidence with intact tissues suggest amino acid transport is mediated by a proton-amino acid symport (L Rheinhold, A Kaplan 1984 Annu Rev Plant Physiol 35: 45-83). However, biochemical studies of proton-coupled amino acid transport in isolated membrane vesicles have not been reported. In the experiments presented here, amino acid t...

The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observ...

An approximate doubling in plant growth, total plant nitrogen, nodulation, and overall dinitrogen fixation of alfalfa are the consequences of the action of a toxin delivered by a Pseudomonas infesting the alfalfa rhizosphere. The toxin, tabtoxinine-beta-lactam, inactivates selectively one form of glutamine synthetase in the nodules. Thus, normal gl...

We report on the effects of administering a unique glutamine synthetase inhibitor to cereals and N/sub 2/-fixing legumes. A bacterium (Pseudomonas syringae pv. tabaci) delivers this inhibitor to provide extended treatment periods; we inoculated the root systems of oat and legume plants with pv. tabaci to provide for delivery of this inhibitor to th...

Tabtoxinine-beta-lactam (T-beta-L), a unique amino acid, is a toxin produced by several closely related pathovars of Pseudomonas syringae. These chlorosis-inducing pathogens establish themselves in the apoplastic space of their hosts where they release the toxin. We have examined the transport of T-beta-L into cultured corn (Zea mays cv Black Mexic...

Tabtoxinine-β-lactam (T-β-L), a unique amino acid, is a toxin produced by several closely related pathovars of Pseudomonas syringae. These chlorosis-inducing pathogens establish themselves in the apoplastic space of their hosts where they release the toxin. We have examined the transport of T-β-L into cultured corn (Zea mays cv Black Mexican) cells...

Inactivation of glutamine synthetase by tabtoxinine-β-lactam was examined in Zea mays cv. Black Mexican suspension culture cells. Tabtoxinine-β-lactam was readily taken up from micromolar solutions of the toxin and, subsequently, rapidly inhibited glutamine synthetase. When cells were treated with 50 μm tabtoxinine-β-lactam, less than 40% of the in...

An extracellular toxin, tabtoxinine-beta-lactam (T beta L), is produced by Pseudomonas syringae pv. "tabaci." This toxin irreversibly inhibits its target, glutamine synthetase; yet P. syringae pv. "tabaci" retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigation of the self-protec...

Tabtoxinine-beta-lactam, an irreversible inhibitor of glutamine synthetase is produced by several pathovars of Pseudomonas syringae. We have examined tabtoxinine-beta-lactam biosynthesis, an important and poorly characterized step in pathogenesis caused by this organism. We have identified the biosynthetic precursors of tabtoxinine-beta-lactam by i...

Glutamine synthetase of plants is the physiological target of tabtoxinine-beta-lactam, a toxin produced by several disease-causing pathovars of Pseudomonas syringae. This toxin, a unique amino acid, is an active site-directed, irreversible inhibitor of glutamine synthetase from pea. ATP is required for inactivation. Neither ADP, AMP, nor adenosine...

The effects of tabtoxinine-beta-lactam (T-beta-L) on nitrate uptake and glutamine synthetase (GS) and nitrate reductase (NR) activities in roots of Avena sativa seedlings were determined. Seven-day-old oat seedlings placed in a 10 mm KNO(3) and 0.5 mm T-beta-L solution for 24 hours took up T-beta-L and lost approximately 90% of their root GS activi...

The effects of tabtoxinine-β-lactam (T-β-L) on nitrate uptake and glutamine synthetase (GS) and nitrate reductase (NR) activities in roots of Avena sativa seedlings were determined. Seven-day-old oat seedlings placed in a 10 mm KNO3 and 0.5 mm T-β-L solution for 24 hours took up T-β-L and lost approximately 90% of their root GS activity. [³H]-T-β-L...

Selected pathovars of Pseudomonas syringae produce an extracellular phytotoxin, tabtoxinine-beta-lactam, that irreversibly inhibits its known physiological target, glutamine synthetase (GS). Pseudomonas syringae subsp. "tabaci" retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigat...

A significant lag phase was observed in the accumulation of product for the reaction catalyzed by pyruvate decarboxylase (PDC) purified from mature maize kernels. The effects of pH, pyruvate, potassium chloride, PDC concentration, and Mg(2+)-thiamine pyrophosphate upon this lag and upon the observed cooperativity were investigated. PDC preincubated...

Pyruvate decarboxylase (PDC) was purified from mature, dry maize kernels and from roots of anaerobically treated maize seedlings and partially characterized. PDC was purified to a specific activity of 96 units per milligram protein from kernels and to 41 units per milligram protein from root. The subunit molecular masses were estimated to be 61,000...

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with Km values of the ord...

The inactivation of glutamine synthetase by tabtoxinine-beta-lactam, a phytotoxin produced by Pseudomonas syringae pv. tabaci, was shown to be irreversible. The chloroplast and cytosolic forms of the enzyme from pea leaves (Pisum sativum L.) were separated, purified, and found to be kinetically similar with K(m) values for glutamate of 6.7 and 4.3...

The inactivation of glutamine synthetase by tabtoxinine-β-lactam, a phytotoxin produced by Pseudomonas syringae pv. tabaci, was shown to be irreversible. The chloroplast and cytosolic forms of the enzyme from pea leaves (Pisum sativum L.) were separated, purified, and found to be kinetically similar with Km values for glutamate of 6.7 and 4.3 milli...

The primary storage protein of oat (Avena sativa L.) seeds, globulin, was shown to have a specific carbohydrate-binding activity. The globulin was capable of hemagglutinating rabbit red blood cells and this hemagglutination was inhibited by the beta-glucan, laminarin, as well as by carbohydrate which had been cleaved from the native globulin. Globu...

Tabtoxinine-beta-lactam, a hydrolytic product of tabtoxin produced by Pseudomonas syringae pv. tabaci, apparently inactivates pea seed glutamine synthetase. Inhibition of the enzyme's initial velocity is linear over a range of 0.5 to 5 millimolar tabtoxinine-beta-lactam in the presence of 10 millimolar glutamate. A method for the purification of gl...

Xylella fastidiosa (Xf), is a gram-negative xylem-limited bacterium and causative agent of Pierce's disease (PD) in California grapevines. During very early stages of Xf infection, specific carbohydrates/lipids/proteins on the outer membrane of Xf interact with plant cells and are important for virulence (3). Design of a protein inhibitor that inte...

Extensive industrial and DOE use of chlorinated hydrocarbons has resulted in widespread soil and ground-water contamination. Bioremediation is a potential remedy because various bacterial strains degrade chlorinated compounds, including trichloroethylene (TCE). Previous reports indicated that the propane monooxygenase (PMO) enzyme from Mycobacteriu...

Carbon emissions and atmospheric concentrations are expected to continue to increase through the next century unless major changes are made in the way carbon is managed. Managing carbon has emerged as a pressing national energy and environmental need that will drive national policies and treaties through the coming decades. Addressing carbon manage...

The authors examined three specific areas relative to COâ emissions and controls: (1) the effect of deregulation of the utility industry on emissions, (2) the role of advanced power systems in reducing emissions, and (3) developing COâ mitigation technologies. In this work the Energy Technologies program office at Los Alamos attempted to initiate a...

Poly(..gamma..-glutamylcysteinyl)glycines, (..gamma..EC) /sub n/G, are functional analogues of metallothionein in Cd-resistant Datura innoxia cells in suspension culture. These peptides bind Cd and the addition of Cd to the growth medium induces their synthesis. The presence of peptide bonds through the ..gamma..-carboxyl group of glutamate indicat...

Improvement of biological nitrogen fixation represents a potential source of both increased food production and decreased dependence on costly chemical fertilizer. They report the results of an investigation of the molecular basis of a unique, microbial-mediated mechanism for increased growth and nitrogen fixation rates in alfalfa. Inoculation of a...

The compound 2-hydroxy-5-oxoproline and analogs thereof may be used to produce an increase in carbon dioxide fixation, growth, dry weight, nutritional value (proteins and amino acids), nodulation and nitrogen fixation and photosynthetically derived chemical energy when applied to plants through their roots and/or through their foliar portions. The...

Manufacture and use of high explosives has resulted in contamination of ground water and soils throughout the world. The use of biological methods for remediation of high explosives contamination has received considerable attention in recent years. Biodegradation is most easily studied using organisms in liquid cultures. Thus, the amount of explosi...

The presence of the pathogen Pseudomonas syringae pv. tabaci within the rhizosphere of nodulated alfalfa plants results in an increase in Nâ-fixation potential and growth, but a 40-50% decrease in nodule glutamine synthetase (GS) activity, as compared to nodulated control plants. Tabtoxinine-..beta..-Lactam an exocellular toxin produced by Pseudomo...

Infestation of the rhizosphere of oat plants with Pseudomonas syringae pv. tabaci results in rapid death of normal oats. This is a consequence of the action of the bacterially delivered inhibitor of glutamine synthetase, tabtoxinine-β-lactam (TβL). Such infested plants contain no active glutamine synthetase. We have screened for a small populatio...

Infestation of legumes such as alfalfa and soybeans with the plant pathogen Pseudomonas syringae pv. tabaci is accompanied by increased plant growth, nodulation, overall nitrogen fixation, and total assimilated nitrogen. These effects are observed only in plants infested with Tox{sup +} pathogen; the toxin is tabtoxinine-β-lactam, an active site-d...

Disaccharides can be prepared enzymatically and by chemical synthesis. Lactose enriched with carbon-13 at C-1 can be synthesized by reacting K¹³CN with a sugar having a one fewer carbon than the desired product. Thus, a mixture of 4-O-..beta..-D-galactopyranosyl-D-(1-¹³C)glucose ((1-¹³C)lactose) and 4-O-..beta..-D-galactopyranosyl-D-(1-¹³C)...