Pentose Phosphate Metabolism Research Articles

An interdomain network: the endobacterium of a mycorrhizal fungus promotes antioxidative responses in both fungal and plant hosts

Arbuscular mycorrhizal fungi (AMF) are obligate plant biotrophs that may contain endobacteria in their cytoplasm. Genome sequencing of Candidatus Glomeribacter gigasporarum revealed a reduced genome and dependence on the fungal host. RNA-seq analysis of the AMF Gigaspora margarita in the presence and absence of the endobacterium indicated that endobacteria have an important role in the fungal pre-symbiotic phase by enhancing fungal bioenergetic capacity. To improve the understanding of fungal-endobacterial interactions, iTRAQ (isobaric tags for relative and absolute quantification) quantitative proteomics was used to identify differentially expressed proteins in G.margarita germinating spores with endobacteria (B+), without endobacteria in the cured line (B-) and after application of the synthetic strigolactone GR24. Proteomic, transcriptomic and biochemical data identified several fungal and bacterial proteins involved in interspecies interactions. Endobacteria influenced fungal growth, calcium signalling and metabolism. The greatest effects were on fungal primary metabolism and respiration, which was 50% higher in B+ than in B-. A shift towards pentose phosphate metabolism was detected in B-. Quantification of carbonylated proteins indicated that the B- line had higher oxidative stress levels, which were also observed in two host plants. This study shows that endobacteria generate a complex interdomain network that affects AMF and fungal-plant interactions.

Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.

The ability to rapidly switch the intracellular energy storage form from starch to lipids is an advantageous trait for microalgae feedstock. To probe this mechanism, we sequenced the 56.8-Mbp genome of Chlorella pyrenoidosa FACHB-9, an industrial production strain for protein, starch, and lipids. The genome exhibits positive selection and gene family expansion in lipid and carbohydrate metabolism and genes related to cell cycle and stress response. Moreover, 10 lipid metabolism genes might be originated from bacteria via horizontal gene transfer. Transcriptomic dynamics tracked via messenger RNA sequencing over six time points during metabolic switch from starch-rich heterotrophy to lipid-rich photoautotrophy revealed that under heterotrophy, genes most strongly expressed were from the tricarboxylic acid cycle, respiratory chain, oxidative phosphorylation, gluconeogenesis, glyoxylate cycle, and amino acid metabolisms, whereas those most down-regulated were from fatty acid and oxidative pentose phosphate metabolism. The shift from heterotrophy into photoautotrophy highlights up-regulation of genes from carbon fixation, photosynthesis, fatty acid biosynthesis, the oxidative pentose phosphate pathway, and starch catabolism, which resulted in a marked redirection of metabolism, where the primary carbon source of glycine is no longer supplied to cell building blocks by the tricarboxylic acid cycle and gluconeogenesis, whereas carbon skeletons from photosynthesis and starch degradation may be directly channeled into fatty acid and protein biosynthesis. By establishing the first genetic transformation in industrial oleaginous C. pyrenoidosa, we further showed that overexpression of an NAD(H) kinase from Arabidopsis (Arabidopsis thaliana) increased cellular lipid content by 110.4%, yet without reducing growth rate. These findings provide a foundation for exploiting the metabolic switch in microalgae for improved photosynthetic production of food and fuels.

Abstract 5168: Tumor metabolism as a driver of lethal prostate cancer

Abstract BACKGROUND: Recent evidence suggests that dysregulation of tumor metabolism is a key feature in the progression of normal prostate epithelium to cancer, and may also reflect disease aggressiveness and metastatic potential. Identifying the altered metabolic processes driving prostate cancer progression is of particular importance in understanding the biology of this disease. METHODS: mRNA was extracted from the tumors of 404 participants of the Health Professionals Follow-up Study and the Physicians’ Health Study diagnosed with incident prostate cancer (113 lethal (prostate cancer death or metastases) and 291 non-lethal cases), and from adjacent histologically normal tissue available for 202 of these cases. mRNA expression was analyzed for 426 metabolic genes encoding enzymes involved in seven metabolic pathways: Fatty Acid metabolism, Glycolysis/Gluconeogenesis, Oxidative Phosphorylation, Pentose Phosphate, Purine metabolism, Pyrimidine metabolism and the TCA cycle. Gene and pathway-level differences in expression between lethal and non-lethal tumors were assessed, and the findings compared to the expression patterns in adjacent normal tissue. Potential confounders and effect modifiers including cohort, age, year of diagnosis, BMI at diagnosis, clinical tumor stage and Gleason grade were explored and multiple testing was accounted for. RESULTS: Two thirds (99/130) of the genes significantly (p<0.05) associated with lethality were overexpressed in the lethal compared to non-lethal tumors, including POLR2K (p = 5.8×10-9), RRM2 (p = 7.7×10-9), ATP6V1A (p = 7.8×10-8) and POLE2 (p = 7.4×10-7). ALDH2 (p = 4.7×10-8) and PDE4DE (p = 7.1×10-8) were the top genes expressed at lower levels in lethal vs. non-lethal cancer. Purine metabolism (p = 5.6×10-9) Pyrimidine metabolism (p = 1.3×10-7) and Oxidative Phosphorylation (p = 5.3×10-6) were the metabolic pathways most strongly upregulated in lethal tumors, while Glycolysis/Gluconeogenesis (p = 8.7×10-12) was inversely associated with lethality. There was no difference in expression of metabolic genes in normal adjacent tissue among the lethal compared to non-lethal cases, however genes in the Pentose Phosphate, Glycolysis/Gluconeogenesis and Fatty acid metabolism pathways were differentially expressed in normal compared to tumor tissue. Results were robust to adjustment for, and stratification by potential confounders including Gleason grade. CONCLUSIONS: These results support the hypothesis that metabolic changes in tumors may have utility in predicting and understanding lethality. A better comprehension of the dysregulation in specific metabolic genes and pathways may offer novel therapeutic targets for the treatment of prostate cancer. Citation Format: Rachel S. Kelly, Jennifer A. Sinnott, Jennifer R. Rider, Ericka Ebot, Travis Gerke, Kathryn Penney, Michaela Bowden, Massimo Loda, Philip W. Kantoff, Neil E. Martin, Edward L. Giovannucci, Andreas Pettersson Pettersson, Svitlana Tyekucheva, Kathryn M. Wilson, Matthew Vander Heiden, Lorelei A. Mucci. Tumor metabolism as a driver of lethal prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5168. doi:10.1158/1538-7445.AM2015-5168

Proteomic analysis of ‘Zaosu’ pear (Pyrus bretschneideri Rehd.) and its early-maturing bud sport

Maturation of fruits involves a series of physiological, biochemical, and organoleptic changes that eventually make fleshy fruits attractive, palatable, and nutritional. In order to understand the mature mechanism of the early-maturing bud sport of ‘Zaosu’ pear, we analyzed the differences of proteome expression between the both pears in different mature stages by the methods of a combination of two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Seventy-five differential expressed protein spots (p<0.05) were obtained between ‘Zaosu’ pear and its early-maturing bud sport, but only sixty-eight were demonstratively identified in the database of NCBI and uniprot. The majority of proteins were linked to metabolism, energy, stress response/defense and cell structure. Additionally, our data confirmed an increase of proteins related to cell-wall modification, oxidative stress and pentose phosphate metabolism and a decrease of proteins related to photosynthesis and glycolysis during the development process of both pears, but all these proteins increased or decreased faster in the early-maturing bud sport. This comparative analysis between both pears showed that these proteins were closely associated with maturation and could provide more detailed characteristics of the maturation process of both pears.

Functional consequences of microbial shifts in the human gastrointestinal tract linked to antibiotic treatment and obesity

The microbiomes in the gastrointestinal tract (GIT) of individuals receiving antibiotics and those in obese subjects undergo compositional shifts, the metabolic effects and linkages of which are not clearly understood. Herein, we set to gain insight into these effects, particularly with regard to carbohydrate metabolism, and to contribute to unravel the underlying mechanisms and consequences for health conditions. We measured the activity level of GIT carbohydrate-active enzymes toward 23 distinct sugars in adults patients (n = 2) receiving 14-d β-lactam therapy and in obese (n = 7) and lean (n = 5) adolescents. We observed that both 14 d antibiotic-treated and obese subjects showed higher and less balanced sugar anabolic capacities, with 40% carbohydrates being preferentially processed as compared with non-treated and lean patients. Metaproteome-wide metabolic reconstructions confirmed that the impaired utilization of sugars propagated throughout the pentose phosphate metabolism, which had adverse consequences for the metabolic status of the GIT microbiota. The results point to an age-independent positive association between GIT glycosidase activity and the body mass index, fasting blood glucose and insulin resistance (r2 ≥ 0.95). Moreover, antibiotics altered the active fraction of enzymes controlling the thickness, composition and consistency of the mucin glycans. Our data and analyses provide biochemical insights into the effects of antibiotic usage on the dynamics of the GIT microbiota and pin-point presumptive links to obesity. The knowledge and the hypotheses generated herein lay a foundation for subsequent, systematic research that will be paramount for the design of “smart” dietary and therapeutic interventions to modulate host-microbe metabolic co-regulation in intestinal homeostasis.

HIV-1 Vpr Modulates Macrophage Metabolic Pathways: A SILAC-Based Quantitative Analysis

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.

A transcriptomic analysis reveals the nature of salinity tolerance of a wheat introgression line

The bread wheat cultivar Shanrong No.3 (SR3) is a salinity tolerant derivative of an asymmetric somatic hybrid between cultivar Jinan 177 (JN177) and tall wheatgrass (Thinopyrum ponticum). To reveal some of the mechanisms underlying its elevated abiotic stress tolerance, both SR3 and JN177 were exposed to iso-osmotic NaCl and PEG stress, and the resulting gene expression was analysed using a customized microarray. Some genes associated with stress response proved to be more highly expressed in SR3 than in JN177 in non-stressed conditions. Its unsaturated fatty acid and flavonoid synthesis ability was also enhanced, and its pentose phosphate metabolism was more active than in JN177. These alterations in part accounted for the observed shift in the homeostasis related to reactive oxygen species (ROS). The specific down-regulation of certain ion transporters after a 0.5 h exposure to 340 mM NaCl demonstrated that Na(+) uptake occurred rapidly, so that the early phase of salinity stress imposes more than simply an osmotic stress. We discussed the possible effect of the introgression of new genetic materials in wheat genome on stress tolerance.

The Reduced Genome of the Parasitic Microsporidian Enterocytozoon bieneusi Lacks Genes for Core Carbon Metabolism

Reduction of various biological processes is a hallmark of the parasitic lifestyle. Generally, the more intimate the association between parasites and hosts the stronger the parasite relies on its host's physiology for survival and reproduction. However, some systems have been held to be indispensable, for example, the core pathways of carbon metabolism that produce energy from sugars. Even the most hardened anaerobes that lack oxidative phosphorylation and the tricarboxylic acid cycle have retained glycolysis and some downstream means to generate ATP. Here we describe the deep-coverage genome resequencing of the pathogenic microsporidiian, Enterocytozoon bieneusi, which shows that this parasite has crossed this line and abandoned complete pathways for the most basic carbon metabolism. Comparing two genome sequence surveys of E. bieneusi to genomic data from four other microsporidia reveals a normal complement of 353 genes representing 30 functional pathways in E. bieneusi, except that only 2 out of 21 genes collectively involved in glycolysis, pentose phosphate, and trehalose metabolism are present. Similarly, no genes encoding proteins involved in the processing of spliceosomal introns were found. Altogether, E. bieneusi appears to have no fully functional pathway to generate ATP from glucose. Therefore, this intracellular parasite relies on transporters to import ATP from its host.

Deuterium NMR study of the origin of hydrogen in fatty acids produced in vivo by chicken

AbstractThe origin of hydrogen in fatty acids produced in vivo by chicken was studied by using isotope tracing monitored by 2H NMR. Chickens were fed with deuterium‐labeled water, deuterium‐labeled glucose/fructose and trideuterated acetate. The site‐specific isotope distribution of the fatty acids produced in vivo by the chickens was analyzed after transformation into methyl esters. A connection was observed between the hydrogen atoms of the feeds and water and those of the different molecular positions of the fatty acids produced by chicken. Water hydrogen makes an important contribution to the formation of the lipids. The hydrogen of the enzyme‐bound acetyl or malonyl moiety in acyl‐CoA may be transferred from the C‐1, C‐2 or C‐6 of glucose or fructose and indirectly from water. The hydrogen introduced by the cofactor NADPH may be abstracted from C‐1 and C‐3 of glucose, through its regeneration in the pentose phosphate metabolism pathway, and contains hydrogen atoms of water. The exogenous acetate enters into the in vivo fatty acid biosynthesis pathway in an efficient way and can make an important contribution to the biosynthesis of the lipids.

Pentose phosphate metabolism in Leishmania mexicana

The metabolism of pentose phosphates was studied in Leishmania mexicana promastigotes. Each of the enzymes of the classical pentose phosphate pathway (PPP) has been identified and specific activities measured. Functioning of the PPP was demonstrated in non-growing cells by measuring the evolution of 14CO2 from [1-14C]D-glucose and [6-14C]D-glucose under normal conditions and also under selective stimulation of the PPP by exposure to methylene blue. The proportion of glucose which passes through the PPP increases in the latter condition, thus suggesting a protective role against oxidant stress. The incorporation into nucleic acids of ribose 5-phosphate provided via either glucose or free ribose was also determined. Results indicate that the PPP enables glucose to serve as a source of ribose 5-phosphate in nucleotide biosynthesis. Moreover, free ribose is incorporated efficiently, implying the presence of a ribose uptake system and also of ribokinase. Ribose was shown to be accumulated by a carrier mediated process in L. mexicana promastigotes and ribokinase activity was also measured in these cells.

Modulation of renal Na-Pi cotransport by hormones acting via genomic mechanism and by metabolic factors

The renal Na-Pi cotransport is subject to multiple regulatory inputs, such as endocrine, paracrine and intracrine. Among lipophilic, long-acting hormones that act via genomic mechanism, thyroid hormones, calcitriol, all-trans-retinoic acid stimulate, whereas glucocorticoids and estradiol inhibit the rate of Na-Pi cotransport across the brush border membrane of proximal tubules in vivo and/or across apical membrane of renal epithelial cells in vitro. Some findings suggest that these hormones may also influence Na-Pi cotransporter by modification of membrane microenvironment. It should be considered that Na-Pi cotransport can be modulated by lipophilic hormones by non-genomic signaling mechanisms such as sphingomyelin-ceramide pathway, NAD-cyclic ADP-ribose-Ca2+i pathway or by Ca2+ influx. Recent studies outline a basis for the putative intracrine signaling mechanisms that utilize Ca(2+)-releasing nucleotides, cyclic ADP-ribose, and nicotinic acid adenosine dinucleotide phosphate (NAADP), as novel second messengers for regulation of Na-Pi cotransport in response to changes of intermediary metabolic processes: gluconeogenesis, pentose phosphate pathway, polyamines and metabolism of fatty acids.

Application of Biochemical Systems Theory to Metabolism in Human Red Blood Cells: SIGNAL PROPAGATION AND ACCURACY OF REPRESENTATION

Human erythrocytes are among the simplest of cells. Many of their enzymes have been characterized kinetically using steady-state methods in vitro, and several investigators have assembled this kinetic information into mathematical models of the integrated system. However, despite its relative simplicity, the integrated behavior of erythrocyte metabolism is still complex and not well understood. Errors will inevitably be encountered in any such model because of this complexity; thus, the construction of an integrative model must be considered an iterative process of assessment and refinement. In a previous study, we selected a recent model of erythrocyte metabolism as our starting point and took it through three stages of model assessment and refinement using systematic strategies provided by biochemical systems theory. At each stage deficiencies were diagnosed, putative remedies were identified, and modifications consistent with existing experimental evidence were incorporated into the working model. In this paper we address two issues: the propagation of biochemical signals within the metabolic network, and the accuracy of kinetic representation. The analysis of signal propagation reveals the importance of glutathione peroxidase, transaldolase, and the concentration of total glutathione in determining systemic behavior. It also reveals a highly amplified diversion of flux between the pathways of pentose phosphate and nucleotide metabolism. In determining the range of accurate representation based on alternative kinetic formalisms we discovered large discrepancies. These were identified with the behavior of the model represented within the Michaelis-Menten formalism. This model fails to exhibit a nominal steady state when the activity of glutathione peroxidase is decreased by as little as 9%. Our current understanding, as embodied in this working model, is in need of further refinement, and the results presented in this paper suggest areas of the model where such effort might profitably be concentrated.

Inhibitory effect of 5-phosphoribosyl 1-pyrophosphate and ADP on the nonoxidative pentose phosphate pathway activity

The rate of the conversion of ribose 5-phosphate to hexose 6-phosphates by reaction of the non-oxidative pentose phosphate pathway was measured in the presence of various biological materials. Of 22 compounds tested, PRPP and ADP markedly inhibited the formation of hexose 6-phosphates from ribose 5-phosphate. The transketolase activity in beef liver enzyme preparation was extremely inhibited by PRPP and ADP, but the transaldolase activity was not inhibited. The mode of inhibition of transketolase by PRPP and ADP was a competitive one. The K i value for PRPP was 0.14 m m and that for ADP 0.54 m m with respect to transketolase. We discuss the possible regulatory roles of ADP and PRPP on pentose phosphate metabolism in the pentose phosphate pathway.

Fosetyl-Al alters the respiratory response in Phytophthora nicotianae var. nicotianae-infected tobacco

Inoculation of susceptible 21-day-old tobacco (cv. 26T68) seedlings by zoospores of the black shank fungus ( Phytophthora nicotianae var. nicotianae) is followed by rapid colonization, cellular degradation and metabolic disruption. Oxygen consumption increases rapidly in the first 13 h after inoculation from 15·3 μmol g −1 h −1 to 31·4 μmol g −1 h −1. By 33 h after inoculation, sporangia have formed profusely, and oxygen consumption rises further to 52·9 μmol g −1 h −1. Up to 12 h after inoculation glycolysis is regulated by pyruvate kinase. After 18 h glycolysis is unregulated. In seedlings protected by 282 μ m fosetyl-Al (aluminium tris ( o-ethylphosphonate)) oxygen consumption rises 7 h after inoculation from a higher initial rate than in untreated seedlings, 19·7 μmol g −1 h −1, to 30·0 μmol g −1 h −1, then falls to 12·9 μmol g −1 h −1 at 15 h. By 33 h after inoculation it has risen again to 37·9 μmol g −1 h −1. This pattern resembles that in a resistant cv., NC 2326. In cv. 26T68 seedlings treated with fosetyl-Al glycolysis remains under the control of phosphofructokinase, and ADP and ATP levels are relatively constant, reflecting only minor disturbances to cellular integrity resulting from the restricted fungal growth. Coinciding with the drop in oxygen consumption between 9 and 15 h after inoculation is a rapid rise in the level of 6-phosphogluconate, indicating a diversion of glycolysis to pentose phosphate metabolism. These observations add to the evidence that changes in the host response to infection are involved in the mode of action of fosetyl-Al.

Citrate regulation of the change in carbohydrate degradation during the initial phase of the citric acid production by Aspergillus niger

A change from pentose phosphate pathway to glycolysis has been shown to occur in the early stages of Aspergillus niger growth in a high citric acid yielding medium. The decrease of pentose phosphate metabolism was shown to be due to inhibition of 6-phosphogluconate dehydrogenase by intracellular citrate. The regulatory enzyme of glycolysis, 6-phosphofructokinase, whose activity could not be detected immediately after spore germination, showed a progressive increase in activity thereafter.

Heterogeneity of isozyme expression in tumor cells does not correlate with metastatic potential.

The major purpose of these studies was to determine whether the expression of isozymes by tumor cells was heterogeneous among tumor cell subpopulations within a neoplasm and whether expression of one or another isozyme correlated with metastatic potential of tumor cells. The expression levels of 40 isozymes were determined in 56 cell lines, many of them clonal, from nine different murine and human tumors. The enzymes chosen for study are involved in nucleotide, carbohydrate and pentose phosphate metabolism, and as such are indicators of the general metabolic and differentiational status of the cell. The tumors studied included two murine and two human malignant melanomas, four murine fibrosarcomas, and one human prostatic adenocarcinoma. The lines isolated from these tumors consisted of cells that are tumorigenic non-metastatic, tumorigenic low metastatic and tumorigenic highly metastatic. Clonally derived cell lines from a given tumor differed in their expression of a number of different isozymes, including adenosine deaminase, creatine phosphokinase-B and lactate dehydrogenase. Different patterns of isozyme expression were observed among different tumor types as well as between tumors of the same type; however, there were no differences in isozyme expression for any enzyme tested that correlated with metastatic ability of tumor cells.

Enzymes of gluconate metabolism and glycolysis in Penicillium notatum.

In addition to the ability of Penicillium notatum to grow on sucrose, glucose, fructose and gluconate, substantial growth occurred on 2-ketogluconate and 5-ketogluconate thereby indicating a diverse sugar metabolism. Cell-free extracts contained all the enzymes of the Embden-Meyerhof-Parnas pathway and for both oxidative and non-oxidative pentose phosphate metabolism. Despite inconsistencies in results between different assay methods for the conventional Entner-Doudoroff (ED) enzymes, the data indicated the route was enzymatically possible. Demonstrations of the activities of the enzymes of the non-phosphorylative equivalent of the ED pathway were achieved. No evidence was found of a phosphorylative linking enzyme between the two pathways. Both 2- and 5-ketogluconate reductases were detected along with gluconate dehydrogenase which suggested interconvertibility between the ketogluconates and gluconate. However, ketogluconokinase, responsible for the conversion of ketogluconate to 2-keto-6-phosphogluconate, was not detected. A scheme for the interrelationships of routes of gluconate metabolism is discussed.

Pentose phosphate metabolism during dormancy breakage in Corylus avellana L.

Commercially obtained fruits of Corylus avellana exhibit the characteristic loss of dormancy of this seed following chilling under moist conditions. The activities of cytosolic and organellar enzymes of pentose phosphate pathway in cotyledonary tissue were assayed throughout stratification and over a similar period in damp vermiculite at 20° C. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconic acid dehydrogenase (6PGDH) were both found in cytosolic extracts in all treatments; only 6PGDH was present in the organellar fraction.The enzyme activities monitored in seeds at 20° C remained relatively constant over the course of the investigation except in the case of cytosolic 6PGDH where it is suggested an inhibitor of the enzyme accumulated. This inhibitor was removed by the partial purification procedure. Increases in the activities of the enzymes occurred during stratification, the major increase coinciding exactly with dormancy breakage but prior to the initiation of germination. The marked increase in G6PDH and 6PGDH concurrent with the change in germination potential of the chilled seed may have considerable biochemical significance in breaking down the dormant state.

Pentose Phosphate Metabolism during Differentiation in Dictyostelium discoideum

Summary: Cell-free extracts of Dictyostelium discoideum contained the enzymes necessary for both oxidative and non-oxidative pentose phosphate metabolism. The specific activities of these enzymes changed little during differentiation. The properties of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were studied with respect to K m values for substrates and cofactor NADP+. The two dehydrogenases were relatively unstable in extracts prepared from early stages of development. The K m value of phospho-glucose isomerase for glucose 6-phosphate was approximately 30-fold higher than that of glucose-6-phosphate dehydrogenase. Measurements of pentose phosphate pathway intermediates were made throughout development. All measured intermediates except fructose 1,6-bisphosphate appeared to accumulate between aggregation and culmination. Fructose 1,6-bisphosphate concentrations remained constant until culmination, then dropped 3-fold during sorocarp construction. Calculation of mass-action ratios for the pentose phosphate reactions suggested that glucose-6-phosphate dehydrogenase was the only reaction greatly displaced from equilibrium. These results are discussed in relation to factors controlling pentose phosphate metabolism during development in D. discoideum.

Biochemical strategy of hepatomas

An insight into the ordered pattern of enzymatic and biochemical imbalance of cancer cells was made possible, at least in part, by the molecular correlation concept, the concept of key enzymes, and the use of biological model systems. With these approaches the pattern of gene expression in neoplasia can be studied in terms of the activities of key enzymes and their linking with neoplastic transformation and progression. An ordered pattern of biochemical imbalance was recognized in carbohydrate, pentose phosphate, purine, pyrimidine, DNA, and polyamine metabolism and in other biochemical areas. Current work is directed to clarifying the enzymology and metabolic pattern of thymidine metabolism and CTP biosynthesis since these areas are of particular importance in selective chemotherapy and rescue. The activities of key enzymes in thymidine metabolism have been correlated with the growth rates in a spectrum of hepatomas. Increases in the activities of four key enzymes in CTP biosynthesis appear to be specific to neoplasia because no similar pattern was observed in the normal adult resting or regenerating liver or in the fetal and developing liver. The overall pattern discovered in transplantable rat hepatomas applies to other rodent tumors. It is of particular importance that the pattern of biochemical imbalance is also applicable to human hepatocellular carcinomas. With the recognition of the ordered pattern of reprogramming of gene expression in hepatomas, the path is open for the development of sensitive assays for biochemical diagnosis of liver tumors and for a rational design of selective chemotherapy and rescue.