Understanding carbamoyl phosphate synthetase deficiency: impact of clinical mutations on enzyme functionality.

Carbamoyl phosphate synthetase I (CPSI) deficiency, a recessively inherited error of the urea cycle, causes life-threatening hyperammonaemia. CPSI is a multidomain 1500-residue liver mitochondrial matrix protein that is allosterically activated by N-acetyl-l-glutamate, and which synthesises carbamoyl phosphate (CP) in three steps: bicarbonate phosphorylation by ATP, carbamate synthesis from carboxyphosphate and ammonia, and carbamate phosphorylation by ATP. Several missense mutations of CPSI have been reported in patients with CPSI deficiency, but the actual pathogenic potential and effects on the enzyme of these mutations remain non-characterised.

Fish macronutrient selection through post-ingestive signals: effect of selective macronutrient deprivation.

Recent reports describe teleosts as being able to regulate energy intake by selecting from pure macronutrient sources, although the regulatory mechanisms involved in this selection remain unknown. The aim of the present work was to determine the effect of selective macronutrient deprivation on energy regulation and macronutrient selection, using for this purpose carbohydrate (CH), protein (P), and fat (F) packaged separately into gelatin capsules, a method that prevents the diet chemosensory properties at oropharyngeal level from interfering with macronutrient selection. Twenty-four individually housed sea bass (Dicentrarchus labrax) were subjected to two experiments: (a) two-macronutrient deprivation, and (b) one-macronutrient deprivation.

N-terminal domain-mediated homodimerization is required for photoreceptor activity of Arabidopsis CRYPTOCHROME 1.

Cryptochromes (CRY) are blue light receptors that share sequence similarity with photolyases, flavoproteins that catalyze the repair of UV light-damaged DNA. Transgenic Arabidopsis thaliana seedlings expressing the C-terminal domains of the Arabidopsis CRY fused to beta-glucuronidase (GUS) display a constitutive photomorphogenic (COP) phenotype, indicating that the signaling mechanism of Arabidopsis CRY is mediated through the C-terminal domain. The role of the Arabidopsis CRY N-terminal photolyase-like domain in CRY action remains poorly understood.

Phy tunes: phosphorylation status and phytochrome-mediated signaling.

Phytochromes are photoreceptors that regulate various aspects of plant growth and development. In this issue of Cell, Ryu et al. (2005) show that PAPP5, a type 5 protein phosphatase, acts on a biologically active phytochrome, increasing its stability and affinity for a downstream signal transducer and thus enhancing plant photoresponses.

An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation.

Tandem affinity purification (TAP) strategies constitute an efficient approach for protein complex purification from many different organisms. However, the application of such strategies for purifying endogenous Arabidopsis multi-protein complexes has not yet been reported. Here, we describe an alternative TAP (TAPa) system that successfully allows protein complex purification from Arabidopsis.

First-time crystallization and preliminary X-ray crystallographic analysis of a bacterial-archaeal type UMP kinase, a key enzyme in microbial pyrimidine biosynthesis.

UMP phosphorylation, a key step for pyrimidine nucleotide biosynthesis, is catalyzed in bacteria by UMP kinase (UMPK), an enzyme specific for UMP that is dissimilar to the eukaryotic UMP/CMP kinase or to other nucleoside monophosphate kinases. UMPK is allosterically regulated and participates in pyrimidine-triggered gene repression. As first step towards determining UMPK structure, the putative UMPK-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus was cloned and overexpressed in Escherichia coli.

Glutamate-5-kinase from Escherichia coli: gene cloning, overexpression, purification and crystallization of the recombinant enzyme and preliminary X-ray studies.

Glutamate-5-kinase (G5K) catalyzes the first step of proline (and, in mammals, ornithine) biosynthesis. It is a key regulatory point of these routes, since it is the subject of feedback allosteric inhibition by proline or ornithine. The Escherichia coli gene (proB) for G5K was cloned in pET22, overexpressed in E.

Arginine biosynthesis in Thermotoga maritima: characterization of the arginine-sensitive N-acetyl-L-glutamate kinase.

To help clarify the control of arginine synthesis in Thermotoga maritima, the putative gene (argB) for N-acetyl-L-glutamate kinase (NAGK) from this microorganism was cloned and overexpressed, and the resulting protein was purified and shown to be a highly thermostable and specific NAGK that is potently and selectively inhibited by arginine. Therefore, NAGK is in T. maritima the feedback control point of arginine synthesis, a process that in this organism involves acetyl group recycling and appears not to involve classical acetylglutamate synthase.

Arabidopsis COP10 forms a complex with DDB1 and DET1 in vivo and enhances the activity of ubiquitin conjugating enzymes.

COP10 is a ubiquitin-conjugating enzyme variant (UEV), which is thought to act together with COP1, DET1, and the COP9 signalosome (CSN) in Arabidopsis to repress photomorphogenesis. Here, we demonstrate that COP10 interacts with ubiquitin-conjugating enzymes (E2s) in vivo, and can enhance their activity in vitro, an activity distinct from previous characterized UEVs such as MMS2 and UEV1. Furthermore, we show that COP10 forms a complex with UV-damaged DNA-binding protein 1a (DDB1a) and de-etiolated 1 (DET1), and physically interacts with COP1 and the CSN.

Oral administration of melatonin reduces food intake and modifies macronutrient selection in European sea bass (Dicentrarchus labrax, L.).

Seasonal and circadian changes have been observed in dietary selection and feeding behavior of fish. It is known that the light-dark cycle is the principal mediator of the production of pineal-derived melatonin in fish, and also that the digestive tract synthesizes and secretes melatonin and indeed is the principal extrapineal source of this secretor product, suggesting that melatonin could be involved in the processes of feeding and/or digestion. Fish are capable of regulating their energy intake from separate sources of macronutrients, but the mechanisms of this selection process are unknown.

Arabidopsis CAND1, an unmodified CUL1-interacting protein, is involved in multiple developmental pathways controlled by ubiquitin/proteasome-mediated protein Degradation.

Ubiquitin/proteasome-mediated protein degradation controls various developmental pathways in eukaryotes. Cullin-containing complexes are both versatile and abundant groups of RING family ubiquitin E3 ligases, whose activities are subject to control by RUB/Nedd8 (for related to ubiquitin/neural precursor cell-expressed developmentally downregulated 8) modification of their cullin subunits. Here, we report the identification of an Arabidopsis thaliana counterpart of human CAND1 (cullin-associated and neddylation-dissociated) and demonstrate that it can preferentially interact with unmodified CUL1.

Interallelic complementation at the Arabidopsis CRE1 locus uncovers independent pathways for the proliferation of vascular initials and canonical cytokinin signalling.

The differentiation of vascular tissue plays a central role in root architecture and its functionality. Regardless of its importance, the molecular mechanisms involved in the inception of vascular morphogenesis and their interaction with hormones are only now beginning to be understood. The characterisation of the WOODEN LEG (wol/cre1 mutant), impaired in procambial cell proliferation and the identification of WOL/CRE1 as a cytokinin receptor, provided the first genetic evidence pointing to a role of cytokinins in the formation of vascular initials.

Molecular phylogenetic studies on the Diatrypaceae based on rDNA-ITS sequences.

The order Diatrypales (Ascomycota) contains one single family, the Diatrypaceae. To obtain insight in the phylogenetic relationships within this family, the complete sequences of the ITS region (ITS1, 5.8S rRNA gene and ITS2) of 53 isolates from the five main genera in the family (Diatrype, Diatrypella, Cryptosphaeria, Eutypa and Eutypella) were determined and aligned for phylogenetic reconstruction.

Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modelling of aspartokinase.

We test, using site-directed mutagenesis, predictions based on the X-ray structure of N-acetyl-L-glutamate kinase (NAGK), the paradigm of the amino acid kinase protein family, about the roles of specific residues on substrate binding and catalysis. The mutations K8R and D162E decreased V([sustrate]= infinity ) 100-fold and 1000-fold, respectively, in agreement with the predictions that K8 catalyzes phosphoryl transfer and D162 organizes the catalytic groups. R66K and N158Q increased selectively K(m)(Asp) three to four orders of magnitude, in agreement with the binding of R66 and N158 to the C(alpha) substituents of NAG.

The COP1-SPA1 interaction defines a critical step in phytochrome A-mediated regulation of HY5 activity.

Arabidopsis COP1 is a constitutive repressor of photomorphogenesis that interacts with photomorphogenesis-promoting factors such as HY5 to promote their proteasome-mediated degradation. SPA1 is a repressor of phytochrome A-mediated responses to far-red light. Here we report that COP1 acts as part of a large protein complex and interacts with SPA1 in a light-dependent manner.

Ex vivo identification, isolation and analysis of tumor-cytolytic T cells.

We isolated pure, viable populations of tumor-cytolytic T cells directly from patient blood samples using flow cytometric quantification of the surface mobilization of CD107a-an integral membrane protein in cytolytic granules-as a marker for degranulation after tumor stimulation. We show that tumor-cytolytic T cells are indeed elicited in patients after cancer vaccination, and that tumor reactivity is strongly correlated with efficient T-cell recognition of peptide-bearing targets. We combined CD107a mobilization with peptide-major histocompatibility complex (P-MHC) tetramer staining to directly correlate antigen specificity and cytolytic ability on a single-cell level.

The course of phosphorus in the reaction of N-acetyl-L-glutamate kinase, determined from the structures of crystalline complexes, including a complex with an AlF(4)(-) transition state mimic.

N-Acetyl-L-glutamate kinase (NAGK), the structural paradigm of the enzymes of the amino acid kinase family, catalyzes the phosphorylation of the gamma-COO(-) group of N-acetyl-L-glutamate (NAG) by ATP. We determine here the crystal structures of NAGK complexes with MgADP, NAG and the transition-state analog AlF(4)(-); with MgADP and NAG; and with ADP and SO(4)(2-). Comparison of these structures with that of the MgAMPPNP-NAG complex allows to delineate three successive steps during phosphoryl transfer: at the beginning, when the attacking and leaving O atoms and the P atom are imperfectly aligned and the distance between the attacking O atom and the P atom is 2.

Characterization of genomic structure and polymorphisms in the human carbamyl phosphate synthetase I gene.

Human carbamyl phosphate synthetase I (CPSI) is an essential hepatic enzyme that initiates the urea cycle. Deficiency of this enzyme usually results in lethal hyperammonemia. CPSI is encoded by the CPSI gene located on chromosome 2q35.

Macronutrient selection through postingestive signals in sea bass fed on gelatine capsules.

The role of the food orosensory properties on protein (P), fat (F) and carbohydrate (CH) self-selection was investigated in fish fed gelatine capsules containing pure macronutrients. A total of 40 sea bass (39.6+/-6.

A phase I trial of SD-9427 (progenipoietin) with a multipeptide vaccine for resected metastatic melanoma.

Purpose: The melanoma tumor antigen epitope peptides MART-1(26-35 (27L)), gp100(209-217 (210M)),and tyrosinase(368-376 (370D)) were emulsified with incomplete Freund's adjuvant and administered with SD-9427 (progenipoietin), an agonist of granulocyte colony-stimulating factor and the FLT-3 receptor, to evaluate the toxicities of and immune responses to this regimen as primary end points and time to relapse and survival as secondary end points.

Experimental Design: Fifteen patients with high-risk resected stage III and IV melanoma were enrolled. Each patient received peptides + incomplete Freund's adjuvant with SD-9427 at doses of either 10, 20, or 40 microg/kg s.

Granulocyte-macrophage-colony-stimulating factor added to a multipeptide vaccine for resected Stage II melanoma.

Background: Forty-eight patients with resected Stages IIA and IIB melanoma were immunized with two tumor antigen epitope peptides derived from gp100(209-217) (210M) (IMDQVPSFV) and tyrosinase(368-376) (370D) (YMDGTMSQV) emulsified with incomplete Freund's adjuvant (IFA). Patients were assigned randomly to receive either peptides/IFA alone or with 250 microm of granulocyte-macrophage-colony-stimulating factor (GM-CSF) subcutaneously daily for 5 days to evaluate the toxicities and immune responses in either arm. Time to recurrence and survival were secondary end points.

High-dose therapy in diffuse large cell lymphoma: results and prognostic factors in 452 patients from the GEL-TAMO Spanish Cooperative Group.

Background: The purpose of this study was to analyse the results and prognostic factors influencing overall survival (OS) and disease-free survival (DFS) in 452 patients diagnosed with diffuse large cell lymphomas (DLCL) treated with high-dose therapy (HDT) included in the Grupo Español de Linfomas/Trasplante Autólogo de Médula Osea (GEL-TAMO) Spanish registry.

Patients And Methods: At transplantation, median age was 42 years (range 15-73), 146 patients (32%) were transplanted in first complete remission (1st CR), 19% in second CR (2nd CR) and 47% had active disease: sensitive disease in 157 (35%) patients [95 were in first partial remission (1st PR) and 62 in second PR (2nd PR)] and refractory disease in 55 (12%) patients. Age-adjusted International Prognostic Index (IPI) was 2 or 3 in 51 patients (12%).

Android fat distribution as predictor of severity in acute pancreatitis.

Background/aims: Obesity is considered an independent risk factor for the development of severe acute pancreatitis (AP). The purpose of this study was to define the type of fat distribution related to severity in AP.

Methods: Eighty-eight patients with first-time AP underwent measurements of weight, height, waist and hip circumferences, and skinfold thickness on admission.