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The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T‐cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance ( CNB1‐1/CNB1 ) abolished FK506–fluconazole synergism. Candida albicans mutants lacking calcineurin B ( cnb1/cnb1 ) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole‐resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.

Eucalypt cultivation is an important economic activity worldwide. In Portugal, Eucalyptus globulus plantations account for one-third of the total forested area. The nutritional requirements of this crop have been well studied, and nitrogen (N) is one of the most important elements required for vegetal growth. N dynamics in soils are influenced by microorganisms, such as diazotrophic bacteria (DB) that are responsible for biological nitrogen fixation (BNF), so the aim of this study was to evaluate and identity the main groups of DB in E. globulus plantations. Samples of soil and root systems were collected in winter and summer from three different Portuguese regions (Penafiel, Gavião and Odemira). We observed that DB communities were affected by season, N fertilization and moisture. Furthermore Bradyrhizobium and Burkholderia were the most prevalent genera in these three regions. This is the first study describing the dynamic of these bacteria in E. globulus plantations, and these data will likely contribute to a better understanding of the nutritional requirements of eucalypt cultivation and associated organic matter turnover.

Aim: New treatments are needed for metastatic UBC. PD-L1 expression is prevalent in UBC and may help protect cancer cells from immune-mediated destruction. MPDL3280A is a human anti-PD-L1 mAb with an engineered Fc-domain which disrupts PD-L1 binding to its receptors PD-1 and B7.1. Methods: In a Ph I study, UBC pts received MPDL3280A 15 mg/kg IV q3w for up to 1 y. Objective response (OR; unconfirmed and confirmed) was assessed by RECIST v1.1. PD-L1 IHC was centrally evaluated on tumor biopsies. Circulating biomarkers were evaluated. Results: As of Jan 1, 2014, 68 UBC pts were treated with MPDL3280A. 67 were evaluable for efficacy (dosed by Nov 20, 2013; with ≥ 6 wk follow-up) and included 30 PD-L1 IHC 2/3, 35 PD-L1 IHC 0/1 and 2 PD-L1 unknown pts. The 67 efficacy evaluable pts were 72% male, had a median age of 65 y (36-86) and 59% were ECOG PS 1. 75% had visceral metastases and 33% had liver metastases. 72% received ≥ 2 prior therapies, 93% received prior platinum-based chemo, 79% received prior cisplatin and 42% received MPDL3280A ≤ 3 mo from last prior chemo. For IHC 2/3 pts, ORR was 43% (95% CI 26-63; 2 CRs, 11 PRs); the median duration of response (DOR) was not reached (range 0.1+ - 30.3+ wk). For IHC 0/1 pts, ORR was 11% (95% CI 4-26; 4 PRs); the median DOR was not reached (range 0.1+ - 6.0+ wk). 16/17 responders were still responding as of clinical cutoff date and included pts with poor prognostic factors. For the 17 responders, the median time to first response was 42 d (38-85 d). The 68 safety-evaluable pts received MPDL3280A for a median duration of 65 d (1-259 d). All grade study drug-related AEs occurred in 57% of pts, most often decreased appetite, fatigue and nausea. Treatment-related G3/4 AEs were seen in 4% of pts (no G4 AEs or treatment-related deaths were observed); there was 1 event each of asthenia, thrombocytopenia and decreased blood phosphorus. Treatment resulted in transient increases in pharmacodynamics biomarkers, including Ki-67 + CD8+ T cells and plasma proteins (eg, IL-18). Updated data will be presented, including PFS. Conclusions: MPDL3280A was well tolerated in this heavily pretreated UBC population. The majority of responses were rapid and on-going, and PD-L1 status correlated with response to MPDL3280A. Disclosure: J. Bellmunt: Genentech consultant, stock; F. Braiteh: has served as a consultant on the speakers bureau for Amgen/Onyxx, Bayer, BMS, Caris Life Sciences, Celgene, Genomic Health, Incyte, INSYS, Novartis, Pfizer, Sanofi and as consultant to Agendia, Foundation Medicine, and Saladax; N. Vogelzang: has served as a consultant for Celgene and Roche/Genentech and has received research funding from Novartis and honoraria from Pfizer; G. Fine, M.S.L. Teng, X. Shen, J. Bruey, Z. Boyd, P. Hegde and D. Chen: is an employee of Genentech, Inc.; Y. Loriot: has served as a consultant for Astellas, Sanofi, Celgene and Pierre Fabre, and received research grants from Astellas and Sanofi. All other authors have declared no conflicts of interest.

5011 Background: Metastatic UBC is associated with a poor prognosis and limited treatment options. PD-L1 expression is prevalent in this disease and may protect cancer cells from immune-mediated de...

3546 Background: This first-in-human study (NCT01577758) assessed the safety and tolerability, dose limiting toxicities (DLTs), maximum tolerated dose (MTD), pharmacokinetics, and preliminary antit...

621^ Background: Preclinical data suggest BEZ235 (BEZ; dual PI3K/mTOR inhibitor) may enhance the activity of PTX ± TZ in pts with advanced cancers. In this 4-arm study (NCT01285466), BEZ or buparli...

5578 Background: In the wake of personalized medicine in Oncology, genetic tumor profiling has become a cornerstone to select patients (pts) for clinical trials, exploring new molecular targeted ag...

Vav works as a GDP/GTP exchange factor for Rac GTPases, thereby facilitating the transition of these proteins from the inactive (GDP-bound) into the active (GTP-bound) state. The stimulation of Vav exchange activity during cell signaling is mediated by tyrosine phosphorylation. To understand the roles of phosphorylation in the regulation of Vav activity, we have initiated the characterization of the residues of Vav that are phosphorylated during signal transduction. Here we show that a Y-to-F mutation in one of these residues, Y174, leads to the oncogenic activation of Vav and to the enhancement of other Vav-mediated signals such as those for cytoskeletal reorganization, JNK activation, and stimulation of the nuclear factor of activated T cells. The effect induced by the Y174F mutation is further accentuated by mutations in residue Y142 or Y160. The Y174F mutation has no effect on the exchange activity of Vav in vitro but results in higher levels of phosphorylation in vivo. Using a phosphospecific antibody, we found that Y174 is phosphorylated following stimulation of mitogenic and antigenic receptors. This phosphorylation event is conserved in Vav-2 and Vav-3, the other two members of the Vav family. These results identify a previously unknown mechanism for the oncogenic activation of Vav and suggest that the activity of this exchange factor is modulated by two antagonistic phosphorylation events, one involved in Vav activation and a second one implicated in Vav inactivation., This work was supported by an NCI grant (1RO1CA7373501) to X.R.B., who is a Sinsheimer Scholar for Cancer Research. The work of C.C. was supported by a fellowship from the Autonomous Government of Catalonia.

Cryptococcus neoformans is a fungal pathogen that causes meningitis in patients immunocompromised by AIDS, chemotherapy, organ transplantation, or high-dose steroids. Current antifungal drug therapies are limited and suffer from toxic side effects and drug resistance. Here, we defined the targets and mechanisms of antifungal action of the immunosuppressant rapamycin in C. neoformans. In the yeast Saccharomyces cerevisiae and in T cells, rapamycin forms complexes with the FKBP12 prolyl isomerase that block cell cycle progression by inhibiting the TOR kinases. We identified the gene encoding a C. neoformans TOR1 homolog. Using a novel two-hybrid screen for rapamycin-dependent TOR-binding proteins, we identified the C. neoformans FKBP12 homolog, encoded by the FRR1 gene. Disruption of the FKBP12 gene conferred rapamycin and FK506 resistance but had no effect on growth, differentiation, or virulence of C. neoformans. Two spontaneous mutations that confer rapamycin resistance alter conserved residues on TOR1 or FKBP12 that are required for FKBP12-rapamycin-TOR1 interactions or FKBP12 stability. Two other spontaneous mutations result from insertion of novel DNA sequences into the FKBP12 gene. Our observations reveal that the antifungal activities of rapamycin and FK506 are mediated via FKBP12 and TOR homologs and that a high proportion of spontaneous mutants in C. neoformans result from insertion of novel DNA sequences, and they suggest that nonimmunosuppressive rapamycin analogs have potential as antifungal agents.

Dissertação apresentada para obtenção do grau de Mestre em Planeamento e Projecto do Ambiente Urbano, nas Faculdades de Arquitectura e de Engenharia da Universidade do Porto, sob a orientação do Prof. Catedrático Nuno Portas

Introduction: PM01183 is a modified analogue of trabectedine currently being evaluated in clinical trials, including germline BRCA mutation carriers. PM01183 binds to the DNA minor groove forming PM01183-DNA adducts that provoke DNA double strand (ds) breaks. The homologous recombination (HR) DNA repair pathway is the major mechanism involved in the repair of these DNA ds breaks, and BRCA mutated cells, which are HR deficient, show great sensitivity to these agents. In BRCA carriers, the somatic loss of the wild type copy of BRCA through loss-of-heterozygosity (LOH) is proposed to be an early event in tumorigenesis. Genomic characterization of somatic BRCA status can yield valuable information for therapeutic decisions and the development of new therapeutic strategies in BRCA carriers. Secondary mutations in BRCA1/2 genes restoring the gene and the HR function have been identified in up to 46% of cisplatin-resistant BRCA-associated ovarian tumors. Secondary mutations have also been identified in breast cancers with acquired resistance to olaparib. Based on the sensitivity of BRCA mutated cells to ds DNA-damaging, we hypothesized that BRCA genetic reversion could appear as one of the mechanism of resistance to PM01183. Methods: Patients with metastatic BRCA-associated breast cancer treated at our institution with PM01183 were enrolled in this study after signing the specifically designed, IRB-approved informed consent form. A blood sample was drawn prior to receiving treatment. Tumor biopsies were performed at baseline and at the time of progression to PM01183 from two patients exhibiting an initial clinical response. Whole exome sequencing (Illumina) was performed on DNA extracted from blood and from fresh-frozen paired tumor samples in order to analyze BRCA1/2 status, BRCA1/2 genetic reversion, and to detect novel genetic events acquired at disease progression. Findings: Sequencing of the BRCA genes in blood confirmed that one patient carried a germline mutation in BRCA1 (c5123C>A), with concomitant LOH found in both tumor samples. The second patient was confirmed a BRCA2-mutation carrier (9610 C>T), without LOH of BRCA2. Secondary mutations in BRCA1/2 genes were not identified in either tumor after progression to PM01183. Other de novo genetic events upon PM01183 progression are under review. Conclusions: Secondary functional-reverting mutations in BRCA1/2 genes have not been identified as a mechanism of resistance to PM01183 in any of the two patients analyzed. LOH of BRCA was not found in the metastatic disease of one patient, thus warranting further investigation of the mechanisms of tumorigenesis in BRCA tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B117. Citation Format: Cristina Cruz, Ana Vivancos, Ginevra Caratu, Yasir Ibrahim, Violeta Serra, Judith Balmana. Analysis of secondary mutations in BRCA1/2 genes as a mechanism of resistance to PM01183 in BRCA-mutation carriers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B117.

Turbot, Scophthlalmus maximus, is a Pleuronectiformes fish that occurs in northeast Atlantic along the European coast and in the Mediterranean Sea and is produced in aquaculture since the last quarter of the twentieth century. During a survey conducted in a turbot fish farm nodular formations were occasionally observed in several organs, especially in the kidney and in the spleen. Microscopic observations showed that these nodules contained acid-fast bacilli. The molecular identification of the isolated bacteria conducted to the Mycobacterium genus. Although no abnormal mortalities were evident morbidity was observed. The normal development and welfare of infected fish decrease and the condition factor, the haematocrit and the haemoglobin concentration in blood decreases significantly with the increase of nodules abundance.

TPS3646 Background: MLN0264, an investigational ADC that targets GCC, consists of a fully human monoclonal antibody conjugated to the cytotoxic microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable linker. MMAE and the linker technology are licensed from Seattle Genetics. GCC is a cell surface protein expressed by normal intestinal epithelial cells, ~95% of metastatic colorectal cancer (mCRC), and subsets of gastric and pancreatic cancers. In normal tissue, GCC is expressed on the apical side of epithelial cell tight junctions; therefore, systemically delivered GCC-targeting agents are expected to be preferentially delivered to tumor tissue in which cell polarity is disrupted. MLN0264 has demonstrated antitumor activity in mouse xenograft models of GCC-expressing tumors (Veiby et al. EORTC-NCI-AACR, 2012), supporting the scientific rationale for this first-in-human study. Methods: This study (NCT01577758) was designed to evaluate MLN0264 in ~60 adult pts with GI malignancies expressing GCC (≥1+), measurable disease by RECIST, and ECOG PS 0/1. This study is being conducted at 4 centers in the US and EU and is estimated to last ~36–42 months. Pts receive IV MLN0264 on day 1 of 21-day cycles for up to 17 cycles or until disease progression/unacceptable MLN0264-related toxicity. Dose escalation is proceeding via an adaptive Bayesian continual reassessment model approach based on dose-limiting toxicities in cycle 1. Following determination of the maximum tolerated dose (MTD), up to 14 pts each will be enrolled to mCRC and gastric carcinoma expansion groups to further characterize the safety, tolerability, and pharmacokinetics (PK) of MLN0264 and evaluate its clinical activity. The primary objectives are to evaluate the safety and tolerability, determine the MTD, and describe the PK of IV MLN0264. Secondary objectives are to evaluate the antitumor activity and immunogenicity of MLN0264. An exploratory objective is to examine the relationship between levels of GCC protein expression in tumor tissue and clinical response. Enrollment as of Jan 2013 was 12 pts. Clinical trial information: NCT01577758.

8008 Background: Human lung cancer expresses high levels of PD-L1, which may inhibit anti-cancer immune responses. MPDL3280A, a human monoclonal Ab containing an engineered Fc-domain designed to optimize efficacy and safety, targets PD-L1, blocking PD-L1 from binding its receptors, including PD-1 and B7.1. Methods: Pts with squamous or nonsquamous NSCLC received MPDL3280A IV q3w at doses between 1-20 mg/kg in a Ph I expansion study. Pts were treated for up to 1 y. Objective response rate (ORR) was assessed by RECIST v1.1. Reported ORR includes u/cCR and u/cPR. Results: As of Jan 10, 2013, 53 NSCLC pts were evaluable for safety and treated at doses of ≤1 (n=2), 10 (n=10), 15 (n=19) and 20 mg/kg (n=22). Pts had a median age of 61 y (range 24-83 y), 98% were PS 0-1, 89% had prior surgery and 55% had prior radiotherapy. 98% of pts received prior systemic therapy. Pts received treatment for a median duration of 106 days (range 1-324) of MPDL3280A. The incidence of all G3/4 AEs, regardless of attribution, was 34%, including pericardial effusion (6%), dehydration (4%), dyspnea (4%) and fatigue (4%). No G3-5 pneumonitis or diarrhea was reported. 37 NSCLC pts enrolled prior to Jul 1, 2012, were evaluable for efficacy. RECIST responses were observed at dose levels between 1 and 20 mg/kg, with all responses ongoing or improving. An ORR of 24% (9/37) was observed in pts with squamous and nonsquamous histologies, including several with rapid tumor shrinkage. Additional pts had delayed responses after apparent radiographic progression (not included in the ORR). The 24-week PFS was 48%. Analysis of biomarker data from archival tumor samples demonstrated a correlation between PD-L1 status and efficacy. Pts who were PD-L1 tumor status–positive showed an ORR of 100% (4/4) and a PD rate of 0% (0/4), while pts who were PD-L1 tumor status–negative showed an ORR of 15% (4/26) and a PD rate of 58% (15/26). Updated data will be presented. Conclusions: Treatment with MPDL3280A was well tolerated, with no pneumonitis-related deaths. Rapid and durable responses were observed. PD-L1 tumor status correlated with response to MPDL3280A. Clinical trial information: NCT01375842.

This paper examines the relative value creation in European listed family firms during the period 2001-2010. Using a sample of 1,325 listed companies, we first replicate previous studies based on a...

16 páginas, 7 figuras., The threatened caesalpinioid legume Dimorphandra wilsonii, which is native to the Cerrado biome in Brazil, was examined for its nodulation and N2-fixing ability, and was compared with another, less-threatened species, D. jorgei. Nodulation and potential N2 fixation was shown on seedlings that had been inoculated singly with five bradyrhizobial isolates from mature D. wilsonii nodules. The infection of D. wilsonii by two of these strains (Dw10.1, Dw12.5) was followed in detail using light and transmission electron microscopy, and was compared with that of D. jorgei by Bradyrhizobium strain SEMIA6099. The roots of D. wilsonii were infected via small transient root hairs at 42 d after inoculation (dai), and nodules were sufficiently mature at 63 dai to express nitrogenase protein. Similar infection and nodule developmental processes were observed in D. jorgei. The bacteroids in mature Dimorphandra nodules were enclosed in plant cell wall material containing a homogalacturonan (pectic) epitope that was recognized by the monoclonal antibody JIM5. Analysis of sequences of their rrs (16S rRNA) genes and their ITS regions showed that the five D. wilsonii strains, although related to SEMIA6099, may constitute five undescribed species of genus Bradyrhizobium, whilst their nodD and nifH gene sequences showed that they formed clearly separated branches from other rhizobial strains. This is the first study to describe in full the N2-fixing symbiotic interaction between defined rhizobial strains and legumes in the sub-family Caesalpinioideae. This information will hopefully assist in the conservation of the threatened species D. wilsonii., This work was partly funded by CAPES grant 2127/09-1 (www.capes.gov.br) and by CSIC grant AGL2008-03360 (www.csic.es), which provided a JAE-predoc scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

3014 Background: Molecular prescreening and biomarker enrichment strategies in Phase 1 trials with targeted therapies are anticipated to improve the outcomes of affected pts. Methods: As part of our personalized oncology program, tumors from pts with advanced chemorefractory CRC were analyzed for specific molecular aberrations (KRAS/ BRAF/ PIK3CA mutations [mut], PTEN and pMET expression) at the Vall d'Hebron Molecular Pathology and Genomics Labs. Those whose tumors were found to have a dysregulation were offered a Phase 1 trial with MTA. Results: During 2010 and 2011, tumor molecular analysis was performed in 254 pts: KRAS mut (80/254, 31.5%), BRAF mut (24/196, 12.2%), PIK3CA mut (15/114, 13.2%), KRAS+PIK3CA mut (9/114, 7.9%), PTEN low (97/183, 53.0% - HSCORE30). In total, 68 pts (median, age 63 yrs; prior therapies 3), received 82 different matched therapies. Type of MTA: PI3K pathway inh (if PIK3CA mut, n=10; or PTEN low, n=32), MEK+PI3K pathway inh (if KRAS mut, n=10; or BRAF mut, n=1), second-generation anti-EGFR mAbs (if KRAS wild-type, n=11), anti-HGF mAb (if pMET high, n=10), mTOR inh + anti-IGFR-1R mAb (if PTEN low, n=5) and BRAF inh (if BRAF mut, n=3). Median time to treatment failure (TTF) with MTA was 7.9 weeks (CI95%:7.6-8.1) vs. 16.3 weeks (CI95%:13.9-18.7) for their prior systemic antitumor therapy (p 16 weeks in 10 cases (12.2%). Clinical benefit, defined as a TTF ratio ≥ 1.3 (TTF on MTA/ TTF on prior therapy), was seen with 15.9% of the therapies (13/82). Conclusions: Preliminary results suggest that matching chemorefractory CRC patients with targeted agents in early clinical trials based on the current molecular profile does not result in longer TTF compared to their prior therapy.

Spinitectus inermis (Zeder, 1800) is a rare specific nematode of the European eel, Anguilla anguilla (L.), that ranges widely in Europe (North, Baltic and Mediterranean Seas basins) but occurs only locally, particularly in lakes, fishponds and in the lowland reaches of rivers (Moravec F. 1977: Folia Parasitol. 24: 85; Moravec F. 1979: Acta Soc. Zool. Bohemoslov. 43: 35-42; Moravec F. 1994: Parasitic Nematodes of Freshwater Fishes of Europe. Academia, Prague, 473 pp.). This nematode has been studied since the beginning of the 19 century but its morphology was little known until the redescription by Moravec (1979, op. cit.) plus additional data revealed by scanning electron microscopy (Moravec F. 1996: Parasite 3: 169-174). The knowledge of the biology of this nematode is also scarce. Recently, however, it was found that nymphs of several species of mayflies (Ephemeroptera) were suitable experimental intermediate hosts of this nematode (Saraiva A.M., Moravec F., Pereira A., Cruz C. 2002: Folia Parasitol. 49: 118-126). Since S. inermis is an abundant parasite of eels in the Sousa River, Portugal, its seasonal occurrence and maturation were followed and the results are presented herein. The population dynamics of S. inermis was followed in eels from the lowest reaches of the Sousa River, a tributary of the Douro River, from the locality of Senhora do Salto, situated about 20 km east of Porto, northern Portugal. Monthly samples were taken by electrofishing from November 1998 to October 1999 (total sample size 326; mean body length of fish 22.0 ± 4.42 cm). Fish were examined for parasites and the recovered nematodes were washed in physiological saline (0.9%), fixed in hot 70% ethanol, stored in 70% ethanol and cleared in glycerol for light microscopical examination for sex and maturity stage determination. The occurrence of nematodes, sex ratio and stages of development of females (gravid females with mature eggs / females in other developmental stages) throughout the year were compared by Chi-square test (Siegel S., Castellan N.J. Jr. 1989: Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill Book Company, New York, 399 pp.). The prevalence and mean intensity of infection of S. inermis in eels during the studied period is shown in Fig. 1. S. inermis was present throughout the study period. Significant difference in the occurrence of this parasite (χ = 32.81, d.f. = 11, p < 0.05) was found during the year. Prevalence reached high values (30-57%) from November to February and low values (10% and 13%) in June and July. Intensity ranged from 1 to 26. The high values of mean intensity occurred in January (9) and June (14), with the minimum value (2) in December. Adults (males and females) of S. inermis occurred in eels Fig. 1. Monthly changes in the prevalence and intensity of Spinitectus inermis in eels from the Sousa River from November 1998 to October 1999. Fish sample size in parentheses.

This paper is part of the contributions to the Proceedings of the 3rd Internacional Symposium on Inorganic Nitrogen Assimilation, held in Tiberias, Israel, 6-11 September, 1992, Most of the nitrate reductase activity (80%;) in carob (Ceratonia siliqua L. cv. Mulata) is localised in the roots. The nitrate concentration in the leaves is relatively low compared to that in the roots, suggesting that nitrate influx into the leaf may be a major factor limiting the levels of nitrate reductase in the shoot. Transport of nitrate from root to shoot appears limited by the entrance of nitrate into the xylem. In order to study this problem, we determined the nitrate concentrations and nitrate reductase activities along the roots of nitrate-grown plants, as well as the composition of the xylem sap and the nitrate levels in the leaves. Some of the the bypocotyl, in order to bypass the loading of nitrate into the xylem of the roots. The results show that the loading of nitrate into the xylem is a limiting step. The cation and anion concentrations of nitrate- and ammonium-fed plants were similar, showing almost no production of organic anions. In both nitrate- and ammonium-fed plants, the transport of nitrogen from root to shoot was in the form of organic nitrogen compounds. The nitrate reductase activity in the roots was more than sufficient to explain all the efflux of OH− into the root medium of nitrate-fed plants. In carob plants the K-shuttle may thus be operative to a limited extent only, corresponding to between 11 and 27%; of the nitrate taken up. Potassium seems to be the cation accompanying stored nitrate in the roots of carob seedlings, since they accumulate nearly stoichiometric amounts of K+ and NO−3.

This paper is part of the contributions to the Proceedings of the 3rd Internacional Symposium on Inorganic Nitrogen Assimilation, held in Tiberias, Israel, 6-11 September, 1992, Seedlings of carob (Ceratonia siliqua L. cv. Mulata) were grown in nutrient solution culture for 5 weeks, with or without nitrogen at different root temperatures (10, 16, 22, 30, 35 or 40deg;C) and with the air temperature kept between 20 and 24°C. The nitrogen was given as either ammonium or nitrate. At all root temperatures studied, nitrogen-depleted plants developed higher net uptake rates for nitrogen than plants grown in the presence of nitrogen. Temperature affected the kinetic parameters of nitrate uptake more than those of ammonium uptake. With increasing root temperature, the Km of ammonium uptake decreased, but to a lesser extent than the Km for nitrate. The increase in Vmax of ammonium uptake with temperature was also less noticeable than that for nitrate uptake. Ammonium and nitrate uptakes were inhibited in a similar way by respiratory or protein synthesis inhibitors. It may be noted that ammonium uptake in the presence of inhibitors at 40°C was higher than uptake at 10°C without inhibitors. Some similarities between the transport mechanisms for nitrate and ammonium are underlined in the present work. Components of both transport systems displayed saturation kinetics and depended on protein synthesis and energy. The following components of nitrate uptake were distinguished: (a) a passive net influx into the apparent free space; (b) a constitutive active uptake and (c) active uptake dependent on protein synthesis. We may similarly define three ammonium uptake systems: (a) a passive influx into the apparent free space; (b) passive diffusion uptake at high temperature and (c) active uptake dependent on protein synthesis. The possible role of the ratio between mechanism (c) and mechanism (b) as determinant of ammonium sensitivity is discussed.

This paper is part of the contributions to the Proceedings of the 3rd International Symposium on Inorganic Nitrogen Assimilation, held in Tiberias, Israel, 6-11 September, 1992, Carob seedlings (Ceratonia siliqua L. cv. Mulata), fed with nitrate or ammonium, were grown in growth chambers containing two levels of CO2 (360 or 800 μl l−1), three root temperatures (15, 20 or 25°C), and the same shoot temperature (20/24°C, night/day temperature). The response of the plants to CO2 enrichment was affected by environmental factors such as the type of inorganic nitrogen in the medium and root temperature. Increasing root temperature enhanced photosynthesis rate more in the presence of nitrate than in the presence of ammonium. Differences in photosynthetic products were also observed between nitrate- and ammonium-fed carob seedlings. Nitrate-grown plants showed an enhanced content of sucrose, while ammonium led to enhanced storage of starch. Increase in root temperature caused an increase in dry mass of the plants of similar proportions in both nitrogen sources. The enhancement of the rates of photosynthesis by CO2 enrichment was proportionally much larger than the resulting increases in dry mass production when nitrate was the nitrogen source. Ammonium was the preferred nitrogen source for carob at both ambient and high CO2 concentrations. The level of photosynthesis of a plant is limited not only by atmospheric CO2 concentration but also by the nutritional and environmental conditions of the root.