De novo and salvage purine synthesis pathways across tissues and tumors.

Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.

Purine nucleotide depletion prompts cell migration by stimulating the serine synthesis pathway.

Purine nucleotides are necessary for various biological processes related to cell proliferation. Despite their importance in DNA and RNA synthesis, cellular signaling, and energy-dependent reactions, the impact of changes in cellular purine levels on cell physiology remains poorly understood. Here, we find that purine depletion stimulates cell migration, despite effective reduction in cell proliferation. Blocking purine synthesis triggers a shunt of glycolytic carbon into the serine synthesis pathway, which is required for the induction of cell migration upon purine depletion. The stimulation of cell migration upon a reduction in intracellular purines required one-carbon metabolism downstream of de novo serine synthesis. Decreased purine abundance and the subsequent increase in serine synthesis triggers an epithelial-mesenchymal transition (EMT) and, in cancer models, promotes metastatic colonization. Thus, reducing the available pool of intracellular purines re-routes metabolic flux from glycolysis into de novo serine synthesis, a metabolic change that stimulates a program of cell migration.

Mitochondrial NADP+ is essential for proline biosynthesis during cell growth.

Nicotinamide adenine dinucleotide phosphate (NADP+) is vital to produce NADPH, a principal supplier of reducing power for biosynthesis of macromolecules and protection against oxidative stress. NADPH exists in separate pools, in both the cytosol and mitochondria; however, the cellular functions of mitochondrial NADPH are incompletely described. Here, we find that decreasing mitochondrial NADP(H) levels through depletion of NAD kinase 2 (NADK2), an enzyme responsible for production of mitochondrial NADP+, renders cells uniquely proline auxotrophic. Cells with NADK2 deletion fail to synthesize proline, due to mitochondrial NADPH deficiency. We uncover the requirement of mitochondrial NADPH and NADK2 activity for the generation of the pyrroline-5-carboxylate metabolite intermediate as the bottleneck step in the proline biosynthesis pathway. Notably, after NADK2 deletion, proline is required to support nucleotide and protein synthesis, making proline essential for the growth and proliferation of NADK2-deficient cells. Thus, we highlight proline auxotrophy in mammalian cells and discover that mitochondrial NADPH is essential to enable proline biosynthesis.

Spliced X-box Binding Protein 1 Stimulates Adaptive Growth Through Activation of mTOR.

BACKGROUND: The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart. METHODS: We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner. RESULTS: We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth. CONCLUSIONS: Our results reveal a critical role of the XBP1s-FKBP11-mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.

Testing the importance of jasmonate signalling in induction of plant defences upon cabbage aphid (Brevicoryne brassicae) attack.

BACKGROUND: Phloem-feeding aphids deprive plants of assimilates, but mostly manage to avoid causing the mechanical tissue damage inflicted by chewing insects. Nevertheless, jasmonate signalling that is induced by infestation is important in mediating resistance to phloem feeders. Aphid attack induces the jasmonic acid signalling pathway, but very little is known about the specific impact jasmonates have on the expression of genes that respond to aphid attack. RESULTS: We have evaluated the function that jasmonates have in regulating Arabidopsis thaliana responses to cabbage aphid (Brevicoryne brassicae) by conducting a large-scale transcriptional analysis of two mutants: aos, which is defective in jasmonate production, and fou2, which constitutively induces jasmonic acid biosynthesis. This analysis enabled us to determine which genes' expression patterns depend on the jasmonic acid signalling pathway. We identified more than 200 genes whose expression in non-challenged plants depended on jasmonate levels and more than 800 genes that responded differently to infestation in aos and fou2 plants than in wt. Several aphid-induced changes were compromised in the aos mutant, particularly genes connected to regulation of transcription, defence responses and redox changes. Due to jasmonate-triggered pre-activation of fou2, its transcriptional profile in non-challenged plants mimicked the induction of defence responses in wt. Additional activation of fou2 upon aphid attack was therefore limited. Insect fitness experiments revealed that the physiological consequences of fou2 mutation contributed to more effective protection against B. brassicae. However, the observed resistance of the fou2 mutant was based on antibiotic rather than feeding deterrent properties of the mutant as indicated by an analysis of aphid feeding behaviour. CONCLUSIONS: Analysis of transcriptional profiles of wt, aos and fou2 plants revealed that the expression of more than 200 genes is dependent on jasmonate status, regardless of external stimuli. Moreover, the aphid-induced response of more than 800 transcripts is regulated by jasmonate signalling. Thus, in plants lacking jasmonates many of the defence-related responses induced by infestation in wt plants are impaired. Constant up-regulation of jasmonate signalling as evident in the fou2 mutant causes reduction in aphid population growth, likely as a result of antibiotic properties of fou2 plants. However, aos mutation does not seem to affect aphid performance when the density of B. brassicae populations on plants is low and aphids are free to move around.

Enantioselective desymmetrization of cyclopropenes by hydroacylation.

We report an enantioselective desymmetrization of cyclopropenes by intermolecular Rh-catalyzed hydroacylation. Cyclopropylketones, bearing quaternary stereocenters, are produced with diastereocontrol (up to >20:1) and excellent enantiomeric excess (up to >99 ee).

Phthalides by rhodium-catalyzed ketone hydroacylation.

Phthalides are biologically relevant five-membered lactones found in herbs, fruits, and vegetables. Herein we communicate the first atom-economical approach to phthalides by using enantioselective ketone hydroacylation. In the presence of Rh[(Duanphos)]X (X = NO(3), OTf, OMs), various 2-ketobenzaldehydes undergo intramolecular hydroacylation to produce phthalide products in good yields and 92-98% ee's. Our study highlights the key role counterions play in controlling both reactivity and enantioselectivity. A concise asymmetric total synthesis of the celery extract (S)-(-)-3-n-butylphthalide is also presented.

Parenting by lying.

The present set of studies identifies the phenomenon of `parenting by lying', in which parents lie to their children as a means of influencing their emotional states and behaviour. In Study 1, undergraduates (n = 127) reported that their parents had lied to them while maintaining a concurrent emphasis on the importance of honesty. In Study 2 (n = 127), parents reported lying to their children and considered doing so to be acceptable under some circumstances, even though they also reported teaching their children that lying is unacceptable. As compared to European American parents, Asian American parents tended to hold a more favourable view of lying to children for the purpose of promoting behavioural compliance.

Kinetics of dendritic cell chimerism and T cell chimerism in allogeneic hematopoietic stem cell recipients.

Dendritic cells (DC) as potent antigen-presenting cells (APC) and T cells as effector cells play an essential role in the pathophysiology of both graft-versus-host (GvH) and graft-versus-leukemia (GvL) reactions after transplantation. Therefore, we determined the kinetics of DC and T-cell chimerism establishment after allogeneic hematopoietic cell transplantation (AHCT) in a group of 144 patients, using fluorescence-activated cell sorting (FACS) or magnetic cell sorting (MACS) followed by FISH or STR-PCR analysis for chimerism evaluation. In all, three cell lines investigated (CD3(+) T cells, CD11c(+) DC1 and CD123(+) DC2), we found a rapid and consistent establishment of complete donor chimerism (CDC) in over 70% of all patients during the first 6 weeks after AHCT. The rate of patients with CDC increased significantly over time within the first year after transplantation. A related donor (P=0.004) as well as an underlying lymphatic leukemia (P=0.03) were found to be significantly associated with development of MC in T cells. No significant correlation between DC or T cell chimerism and GvHD or relapse was detected. Our results thus demonstrate a fast and stable CDC in DC1, DC2 and T cells after AHCT that continuously increases over time in nearly all patients.

Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission--experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG).

Patients with CLL responding to initial chemotherapy with fludarabine alone (F) or in combination with cyclophosphamide (FC) were randomized for treatment with alemtuzumab (30 mg i.v. TIW, 12 weeks) or observation. Of 21 evaluable patients, 11 were randomized to alemtuzumab before the study was stopped due to severe infections in seven of 11 patients. These infections (one life-threatening pulmonary aspergillosis IV; four CMV reactivations III requiring i.v. ganciclovir; one pulmonary tuberculosis III; one herpes zoster III) were successfully treated and not associated with cumulative dose of alemtuzumab. In the observation arm, one herpes zoster infection II and one sinusitis I were documented. At 6 months after randomization, two patients in the alemtuzumab arm converted to CR, while three patients in the observation arm progressed. After alemtuzumab treatment, five of six patients achieved a molecular remission in peripheral blood while all patients in the observation arm remained MRD-positive (P=0.048). At 21.4 months median follow-up, patients receiving alemtuzumab showed a significant longer progression-free survival (no progression vs mean 24.7 months; P=0.036). In conclusion, a consolidation therapy with alemtuzumab is able to achieve molecular remissions and longer survival in CLL, but a safe treatment regimen needs to be determined.

Does ethylene mediate cluster root formation under iron deficiency?

Casuarina glauca develops proteoid (cluster) roots in response to Fe deficiency. This study set out to investigate the possible involvement of ethylene in the initiation and/or the morphogenesis of cluster roots (CR). For this purpose, the effect of Ag+ added as silver thiosulfate, an inhibitor of ethylene action has been studied in plants growing hydroponically. No CR formation was observed in these growth conditions. Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine, 1- aminoisobutyric acid, aminoxyacetic acid or cobalt chloride also eliminated the positive effect of Fe deficiency on CR formation in C. glauca. CR were not formed in Fe- deficient roots in the presence of ethylene inhibitors, suggesting a role for ethylene in the morphological responses to Fe deficiency. Interestingly, treatment of Casuarina plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid stimulated significantly the formation of CR, even if plants are supplied with Fe. However, this stimulation did not reach the level of CR obtained in Fe-deficient plants. These results suggest that an ethylene-mediated signalling pathway is involved in CR formation process in C. glauca.

Pediatric liver transplantation for biliary atresia: results of primary grafts in 328 recipients.

PURPOSE: The purpose of this study was to assess the overall results of recipients undergoing transplantation for biliary atresia (BA), according to age, surgical techniques, and transplant eras, and to identify the prognostic factors affecting outcome. METHODS: Between 1984 and 2000, 328 pediatric recipients with BA who underwent orthotopic liver transplantation (OLT) were reviewed. Median age at OLT was 1.5 years (range, 0.4-14.5 years). Kasai hepatoportoenterostomy (KHPE) had been previously performed in 285 (87%) children. Regarding surgical techniques, 125 (38%) children received a whole-liver graft, 128 (39%) received a reduced-size graft, 16 (5%) received a split-liver graft, and 59 (18%) received a living-related (LR) donor graft. RESULTS: Overall actuarial patient survivals were 87%, 83%, and 81% at 1, 5, and 10 years, respectively. One-year patient survivals in children undergoing transplantation at the different age ranges were 85% (under 1 year), 86% (1-3 years), 83% (3-6 years), 100% (6-10 years), and 100% (beyond 10 years) (not significant). One-year patient survivals for the different transplant eras were 75% (1984-1988), 85% (1989-1992), 93% (1993-1996), and 98% (1997-2000) (P=0.0001). Multivariate analysis demonstrated that pretransplant recipient weight (P=0.004), indication for OLT (P=0.083), and age at OLT (P=0.024) predicted patient survival. The type of baseline calcineurin inhibitor (tacrolimus) and the age at OLT (beyond 6 years) were significantly associated with a better graft survival. CONCLUSIONS: Best results in children undergoing transplantation beyond 6 years indicate the importance of performing a KHPE as the first therapeutic step in BA; innovative surgical techniques, particularly LR donor graft, allowed successful transplantation in infants with early failure of KHPE.

[Persistent polyclonal B-cell lymphocytosis]. / Persistierende polyklonale B-Zell-Lymphozytose.

HISTORY AND CLINICAL FINDINGS: A 54-year-old woman was referred for ambulant checkup after an episode of acute renal failure due to severe gastroenteritis and recurrent arthralgias. Physical examination was unremarkable except for the presence of palpable small cervical lymph nodes. INVESTIGATIONS: Serum IgM levels showed a polyclonal increase. All the other routinely examined parameters were within normal limits. Microscopical blood smear examination revealed binucleated lymphocytes. Immunophenotyping of peripheral blood showed a polyclonal B-cell lymphocytosis despite normal numbers of leukocytes and lymphocytes. PCR analysis identified cells with a t(14;18) translocation (bcl-2/IgH rearrangement). DIAGNOSIS: A routine medical checkup disclosed the diagnosis of persistent polyclonal B-cell lymphocytosis. This rare benign lymphoproliferative disorder is characterized by binucleated lymphocytes, polyclonal expansion of B-cells, and a polyclonal increase in serum IgM. The diagnosis was established despite the lack of leukocytosis or lymphocytosis in the peripheral blood. CONCLUSIONS: Because of its benign and indolent course without the need for chemotherapy, it is important to discriminate the disorder of persistent polyclonal B-cell lymphocytosis from other malignant lymphoproliferative diseases.

Performance of the XE-2100 leucocyte differential.

The XE-2100 trade mark was evaluated in a multicentre study following a previously established protocol. In this paper, we demonstrate the results of analytical performance studies, including comparison of the leucocyte differential with the NCCLS H20-A method and evaluation of flagging sensitivity. Linearity of the leucocyte count over a wide clinical range, low imprecision in clinically important ranges and no measurable carry over were confirmed. For comparability studies, 4 x 200 cell microscopic differential leucocyte counts were correlated with the automated five-part-differential counts. No significant differences were detected in (1) a group without morphological abnormality and in (2) a leukopenic group. The sensitivity of flags for the detection of immature granulocytes and myeloid blasts was very good. Only few samples containing blast cells remained unrecognized but these would have been examined microscopically in any event because of other abnormalities indicated by the instrument. Atypical/abnormal lymphocytes/and lymphoblasts were detected very reliably when the total lymphocyte count and the flags were evaluated in combination. A similar procedure is recommended for the detection of left shift. When the neutrophil count is elevated, the sensitivity of the left shift flag is improved. The absolute immature granulocyte (IG) count by the instrument correlates well with that of myeloid precursor cells by microscopy.

Cryopreservation of reduced cytochrome C for determination of N-formyl-methionyl-leucyl-phenylalanine-stimulated superoxide anion production in human whole blood.

Various methods are available for measuring the production of reactive oxygen species by phagocytes, but they are limited in their use by the need for their immediate application, cell isolation and of cell-activation by unphysiological stimuli. In addition, after measurement of reactive oxygen metabolites using oxidizing agents, the reduced compounds formed have to be determined during or immediately after their formation. In the present study, an improved cytochrome C assay was investigated which allowed measurements of superoxide anions in whole blood samples following activation of phagocytes by physiological stimuli such as the bacterial tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). The fMLP-stimulated production of superoxide anion (O(2)(-)) showed a sigmoidal-shaped fMLP dose-response curve, and constant O(2)(-) production rates (nmol.1(-1)x10(6) granulocytes) could be determined reliably up to a blood granulocyte concentration of 1 x 10(4) x microl(-1). To allow the determination of reduced cytochrome C later after its formation, the effect of long-term storage at -20 degrees C on the stability of reduced cytochrome C was tested up to 16 weeks. The results obtained show that the determination of reduced cytochrome C in whole blood represents a simple and reliable method. Most importantly, O(2)(-)-reduced cytochrome C can be frozen and stored without any alterations, at least up to 2 weeks. Thus the method seems to be superior to other methods of detection, especially when the experimental conditions do not allow immediate spectrophotometry (e.g. mountain medicine, space medicine). Under such conditions the present assay would allow reliable measurement of reduced cytochrome C, even after weeks of cryopreservation.

Levels of insulin-like growth factor after stem cell transplantation.

Some cytokines, i.e. tumor necrosis factor-, interleukin-6 and soluble interleukin-2 receptors are associated with complications of stem cell transplantation. Insulin-like growth factors (IGFs) are a family of peptides essential for the proliferation of normal and malignant cells. Recently increased levels of IGFs have been associated with the development of malignant tumors. In this communication we report on 96 measurements of insulin-like growth factor-I (IGF-1), insulin-like growth factor-II (IGF-2), and insulin-like growth factor-binding protein-3 (IGFBP-3) performed in 19 patients following stem cell transplants. Seventeen patients had allogeneic and 2 patients autologous transplants. Most IGF determinations were made at days 0, 7, 14, 21 and 28, some at other time points. The baseline values (day 0) of IGF-1 and IGFBP-3 were not different from controls. IGF-2 values were slightly lower than controls. Following transplantation, a consistent increase of IGF-1 was observed in 9/16 patients at days 7 and 14. Later the values decreased again. IGF-2 and IGFBP-3 did not change significantly after transplantation. No direct correlation could be established with the severity of graft-versus-host disease, levels of interleukin-6 and the time to hematopoietic recovery. A potential relevance of IGFs following stem cell transplantation may be the early diagnosis of liver damage and the development of second malignancies. More studies are necessary to investigate the pathophysiology and the clinical relevance of the increase of IGF-1 following stem cell transplantation.

Angiosperm Gymnostoma trees produce root nodules colonized by arbuscular mycorrhizal fungi related to Glomus.

• Structure and fungal composition is presented here for 'mycorrhizal' nodules of two angiosperms of the genus Gymnostoma (Casuarinaceae), G. deplancheanum and G. nodiflorum. These species are endemic to New Caledonia, where they grow on ultramafic soils. The mycorrhizal nodules, which are modified lateral roots invaded by an arbuscular mycorrhizal fungus, occur in addition to N2 -fixing nodules. • Techniques included PCR amplification of extracted DNA, for species identification, and histological studies to compare the developmental pathway of Gymnostoma mycorrhizal nodules with that of actinorhizal nodules. • The fungal DNA suggested that the strain belongs to the genus Glomus (Glomales). The endophytic mycelium also contained typical Glomus arbuscules and hyphal coils. Structurally, Gymnostoma mycorrhizal nodules are similar to those described in some Coniferales and in Caesalpinioideae trees of French Guyana. • The mycorrhizal nodules of G. deplancheanum and G. nodiflorum contain a fungus belonging to the Glomales. The role of the nodules might be linked to the ecological situation of the host plants, which are pioneers in exposed and rocky habitats.

Expression and functional activity of P-glycoprotein in adult acute myelogenous leukemia patients.

Expression and functional activity of P-glycoprotein (P-gp) were measured in 182 acute myelogenous leukemia (AML) patients: 136 patients were treated with the AML-6 protocol (EORTC), containing daunorubicin, vincristine, and conventional-dose cytarabine (ara-C), and 21 patients received idarubicin, vepeside, and conventional-dose ara-C (ICE-AML-10 protocol/EORTC). An additional 25 patients were treated with a dose of idarubicin and ara-C, modified as compared with the ICE protocol, but with the same dose of etopside (ICE-I protocol). P-gp was determined using monoclonal antibody 4E3.16 and functional activity using the rhodamine 123 accumulation test. P-gp positivity was defined as a Kolmogorov Smirnov (KS) D value > or = 0.15, P-gp negativity as a KS D value < 0.15. P-gp activity was defined as a ratio of mean rhodamine 123 accumulation with/without verapamil. In AML patients at primary diagnosis and early relapse/refractoriness a significant (p < 0.05) difference between P-gp-positive and P-gp-negative patients was ascertained using the AML-6 protocol; the difference corresponded to the complete remission rate. For ICE- and ICE-I-treated AML patients at primary diagnosis this significance was not shown. Compared with AML patients at primary diagnosis and patients at early relapse or refractoriness, a significantly (p < 0.05) increased incidence of non-pumping P-gp and a trend (p = 0.054) to a higher percentage of non-P-gp-related mechanisms in AML patients at late relapse was determined. When the AML-6 protocol is used, age, activated P-gp, and CD34 expression are independent prognostic factors in AML patients. A test system which determines a functional P-gp overexpression is a major tool for identifying a group of AML patients with a poor prognosis. In order to effectively use so-called P-gp modulator substances, the degree of P-gp expression, the activated or nonactivated P-gp condition, and detection of non-P-gp-related resistance mechanisms are of utmost interest for optimal design and analysis of P-gp modulator trials and for understanding the complexity of chemotherapy-related resistance mechanisms in patients.