Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules

NADH-dependent glutamate synthase (NADH-GOGAT) is a key enzyme in primary ammonia assimilation in Phaseolus vulgaris nodules. Two different types of cDNA clones of PvNADH-GOGAT were isolated from the nodule cDNA libraries. The full-length cDNA clones of PvNADH-GOGAT-I (7.4 kb) and PvNADH-GOGAT-II (7.0 kb), which displayed an 83% homology between them, were isolated using cDNA library screening, 'cDNA library walking' and RT-PCR amplification. Southern analysis employing specific 5' cDNA probes derived from PvNADH-GOGAT-I and PvNADH-GOGAT-II indicated the existence of a single copy of each gene in the bean genome. Both these proteins contain ∼100 amino acid sequences theoretically addressing each isoenzyme to different subcellular compartments. RT-PCR analysis indicated that PvNADH-GOGAT-II expression is higher than PvNADH-GOGAT-I during nodule development. Expression analysis by RT-PCR also revealed that both of these genes are differentially regulated by sucrose. On the other hand, the expression of PvNADH-GOGAT-I , but not PvNADH-GOGAT-II, was inhibited with nitrogen compounds. In situ hybridization and promoter expression analyses demonstrated that the NADH-GOGAT-I and -II genes are differentially expressed in bean root and nodule tissues. In silico analyses of the NADH-GOGAT promoters revealed the presence of potential cis elements in them that could mediate differential tissue-specific, and sugar and amino acid responsive expression of these genes.     

Purification and Characterization of an Extracellular Non-Aspartyl Acid Protease (pumAe) from <Emphasis Type="Italic">Ustilago maydis</Emphasis>

The proteinase pumAe was purified to homogeneity from haploid U. maydis FB1 growing on acid mineral medium. The purification procedure consisted of ammonium sulfate fractionation and gel filtration chromatography, resulting in a 7.7% recovery and a 15.1-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 72 kDa and 74 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 4.0 and at 45°C toward hemoglobin, and the pI was determined to be 5.5. The effects of six protease inhibitors on pumAe were tested, and no inhibitory effect was observed. The pure enzyme degraded gelatin and albumin, but casein and collagen were not degraded. The K_m value was 3.5 M, and the V_max value was 11430 mol h^–1 mg^–1 for Suc-R-P-F-H-L-L-V-Y-MCA.     

A Phase I,Dose-Finding Study of Sorafenib in Combination with Gemcitabine and Radiation Therapy in Patients with Unresectable Pancreatic Adenocarcinoma: A Grupo Espa?ol Multidisciplinario en Cáncer Digestivo (GEMCAD) Study

Purpose

Sorafenib, an oral inhibitor of B-raf, VEGFR2, and PDGFR2-beta, acts against pancreatic cancer in preclinical models. Due to the radio-sensitization activity of both sorafenib and gemcitabine, we designed a multicenter, phase I trial to evaluate the safety profile and the recommended dose of this combination used with concomitant radiation therapy.

Methods

Patients with biopsy-proven, unresectable pancreatic adenocarcinoma (based on vascular invasion detected by computed tomography) were treated with gemcitabine (300 mg/m2 i.v. weekly ×5 weeks) concurrently with radiation therapy (45 Gy in 25 fractions) and sorafenib (escalated doses in a 3+3 design, from 200 to 800 mg/day). Radiation portals included the primary tumor but not the regional lymph nodes. Patients with planning target volumes (PTV) over 500 cc were excluded. Cases not progressing during chemoradiation were allowed to continue with sorafenib until disease progression.

Results

Twelve patients were included. Three patients received 200 mg/day, 6 received 400 mg/day, and 3 received 800 mg/day; PTVs ranged from 105 to 500 cc. No dose-limiting toxicities occurred. The most common grade 2 toxicities were fatigue, neutropenia, nausea, and raised serum transaminases. Treatment was discontinued in one patient because of a reversible posterior leukoencephalopathy. There were no treatment-related deaths.

Conclusion

The addition of sorafenib to concurrent gemcitabine and radiation therapy showed a favorable safety profile in unresectable pancreatic adenocarcinoma. A dose of 800 mg/day is recommended for phase II evaluation.

Trial Registration

EudraCT 2007-003211-31 ClinicalTrials.gov 00789763     

Differential accumulation of mRNAs in drought-tolerant and susceptible common bean cultivars in response to water deficit

The physiological response to drought was measured in two common bean varieties with contrastive susceptibility to drought stress. A subtractive cDNA library was constructed from the two cultivars, Phaseolus vulgaris'Pinto Villa' (tolerant) and 'Carioca' (susceptible). 18 cDNAs displayed protein-coding genes associated with drought, cold and oxidative stress, signal transduction, plant defense, chloroplast function and unknown function. A cDNA coding for an aquaporin (AQP) was selected for further analyses. The open reading frames (ORFs) of AQPs from 'Pinto Villa' and 'Carioca' were compared and despite their similarity, accumulated differentially in the plant organs, as demonstrated by Northern blot and in situ hybridization. A phylogenetic analysis of the deduced amino acid sequence with other AQPs suggested a tonoplast-located protein. Under drought conditions, the levels of AQP mRNA from the susceptible cultivar decreased to undetectable levels; by contrast, 'Pinto Villa' mRNA was present and restricted the phloem tissue. This would allow 'Pinto Villa' to maintain vascular tissue functions under drought stress.     

Schistosoma mansoni: In vitro schistosomicidal activity of essential oil of Baccharis trimera (less) DC

Schistosomiasis is a chronic parasitic disease caused by the trematode species Schistosoma mansoni. Chemotherapy is the only immediate recourse to minimize the prevalence and incidence of this disease worldwide. At present, praziquantel (PZQ) is the drug of choice for the treatment of all forms of schistosomiasis. However, dependence on a single drug is concern because some strains can become resistant. In this context, medicinal plants become potential candidates as sources of new drug prototypes. This study provides findings on the schistosomicidal activity of the essential oil of Baccharis trimera in in vitro assays. During the assays parameters such as motility of adult worms, oviposition, morphological changes on the tegument and especially the mortality rate of adult worms of the BH strain were evaluated. The assays, which were carried out with four concentrations - 24, 48, 91 and 130μg/mL - of the essential oil, have shown a promising activity regarding the parameters under study. It was possible to notice a significant decline in the motility of the worms and a mortality rate of 100% 30h after they had been exposed to the essential oil in the concentration of 130μg/mL. Male worms were more susceptible, producing a dose-response effect within a smaller exposition period than female worms. In what refers to morphological changes, the essential oil of B. trimera induced a peeling on the tegument surface, as well as the destruction of tubercles and spines, which resulted in smooth areas on the body surface. The essential oil also caused tegument destruction in female worms, in addition to destruction of the oral and acetabular suckers. It is the first time that the schistosomicidal activity has been reported for essential oil of B. trimera (less) DC.     

Mutation analysis of phenylketonuria in Spain: prevalence of two Mediterranean mutations

Summary We have screened seven Spanish phenylketonuric (PKU) families for the most prevalent Mediterranean and Caucasian mutations, and have subsequently found mutations P281L and IVS10. We have analyzed these two mutations in 23 of our patients. The frequencies found correspond to those of Mediterranean countries, such as Italy. This is the first report of a mutation analysis in the Spanish PKU population.     

A Quantitative Assessment of the Role of the Parasite Amoebophrya in the Termination of Alexandrium fundyense Blooms within a Small Coastal Embayment

Parasitic dinoflagellates of the genus Amoebophrya infect free-living dinoflagellates, some of which can cause harmful algal blooms (HABs). High prevalence of Amoebophrya spp. has been linked to the decline of some HABs in marine systems. The objective of this study was to evaluate the impact of Amoebophrya spp. on the dynamics of dinoflagellate blooms in Salt Pond (MA, USA), particularly the harmful species Alexandrium fundyense. The abundance of Amoebophrya life stages was estimated 3–7 days per week through the full duration of an annual A. fundyense bloom using fluorescence in situ hybridization coupled with tyramide signal amplification (FISH- TSA). More than 20 potential hosts were recorded including Dinophysis spp., Protoperidinium spp. and Gonyaulax spp., but the only dinoflagellate cells infected by Amoebophrya spp. during the sampling period were A. fundyense. Maximum A. fundyense concentration co-occurred with an increase of infected hosts, followed by a massive release of Amoebophrya dinospores in the water column. On average, Amoebophrya spp. infected and killed ∼30% of the A. fundyense population per day in the end phase of the bloom. The decline of the host A. fundyense population coincided with a dramatic life-cycle transition from vegetative division to sexual fusion. This transition occurred after maximum infected host concentrations and before peak infection percentages were observed, suggesting that most A. fundyense escaped parasite infection through sexual fusion. The results of this work highlight the importance of high frequency sampling of both parasite and host populations to accurately assess the impact of parasites on natural plankton assemblages.