A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708–831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.

Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: A model for viral DNA binding

Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us to generate multidomain HIV-1 IN crystals. The first multidomain HIV-1 IN structure is reported. It incorporates the catalytic core and C-terminal domains (residues 52–288). The structure resolved to 2.8 Å is a Y-shaped dimer. Within the dimer, the catalytic core domains form the only dimer interface, and the C-terminal domains are located 55 Å apart. A 26-aa α-helix, α6, links the C-terminal domain to the catalytic core. A kink in one of the two α6 helices occurs near a known proteolytic site, suggesting that it may act as a flexible elbow to reorient the domains during the integration process. Two proteins that bind DNA in a sequence-independent manner are structurally homologous to the HIV-1 IN C-terminal domain, suggesting a similar protein–DNA interaction in which the IN C-terminal domain may serve to bind, bend, and orient viral DNA during integration. A strip of positively charged amino acids contributed by both monomers emerges from each active site of the dimer, suggesting a minimally dimeric platform for binding each viral DNA end. The crystal structure of the isolated catalytic core domain (residues 52–210), independently determined at 1.6-Å resolution, is identical to the core domain within the two-domain 52–288 structure.

Eotaxin is required for the baseline level of tissue eosinophils

Eotaxin is an eosinophil-selective chemokine that is constitutively expressed in a variety of organs such as the intestine. Previous studies have demonstrated that the recruitment of eosinophils during inflammation is partially dependent on eotaxin, but the function of constitutive eotaxin during homeostasis has not been examined. To elucidate the biological role of this molecule, we now examine tissue levels of eosinophils in healthy states in wild-type and eotaxin-deficient mice. The lamina propria of the jejunum of wild-type mice is demonstrated to express eotaxin mRNA, but not mRNA for the related monocyte chemoattractant proteins. Wild-type mice contained readily detectable eosinophils in the lamina propria of the jejunum. In contrast, mice genetically deficient in eotaxin had a large selective reduction in the number of eosinophils residing in the jejunum. The reduction of tissue eosinophils was not limited to the jejunum, because a loss of thymic eosinophils was also observed in eotaxin-deficient mice. These studies demonstrate that eotaxin is a fundamental regulator of the physiological trafficking of eosinophils during healthy states. Because a variety of chemokines are constitutively expressed, their involvement in the baseline trafficking of leukocytes into nonhematopoietic tissue should now be considered.

The unpredictability paradox: review of empirical comparisons of randomised and non-randomised clinical trials

Objective To summarise comparisons of randomised clinical trials and non-randomised clinical trials, trials with adequately concealed random allocation versus inadequately concealed random allocation, and high quality trials versus low quality trials where the effect of randomisation could not be separated from the effects of other methodological manoeuvres.

The Medicago Genome Initiative: a model legume database

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume Medicago truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated Medicago functional genomics program at the Noble Foundation (http://www.noble .org), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive Gαq coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2–16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.

The Role of EDTA in Lead Transport and Accumulation by Indian Mustard1

Indian mustard (Brassica juncea) plants exposed to Pb and EDTA in hydroponic solution were able to accumulate up to 55 mmol kg−1 Pb in dry shoot tissue (1.1% [w/w]). This represents a 75-fold concentration of Pb in shoot tissue over that in solution. A threshold concentration of EDTA (0.25 mm) was found to be required to stimulate this dramatic accumulation of both Pb and EDTA in shoots. Below this threshold concentration, EDTA also accumulated in shoots but at a reduced rate. Direct measurement of a complex of Pb and EDTA (Pb-EDTA) in xylem exudate of Indian mustard confirmed that the majority of Pb in these plants is transported in coordination with EDTA. The accumulation of EDTA in shoot tissue was also observed to be directly correlated with the accumulation of Pb. Exposure of Indian mustard to high concentrations of Pb and EDTA caused reductions in both the transpiration rate and the shoot water content. The onset of these symptoms was correlated with the presence of free protonated EDTA (H-EDTA) in the hydroponic solution, suggesting that free H-EDTA is more phytotoxic than Pb-EDTA. These studies clearly demonstrate that coordination of Pb transport by EDTA enhances the mobility within the plants of this otherwise insoluble metal ion, allowing plants to accumulate high concentrations of Pb in shoots. The finding that both H-EDTA and Pb-EDTA are mobile within plants also has important implications for the use of metal chelates in plant nutritional research.

Dimethylsulfoniopropionate Biosynthesis in Spartina alterniflora1 : Evidence That S-Methylmethionine and Dimethylsulfoniopropylamine Are Intermediates

The osmoprotectant 3-dimethylsulfoniopropionate (DMSP) occurs in Gramineae and Compositae, but its synthesis has been studied only in the latter. The DMSP synthesis pathway was therefore investigated in the salt marsh grass Spartina alterniflora Loisel. Leaf tissue metabolized supplied [35S]methionine (Met) to S-methyl-l-Met (SMM), 3-dimethylsulfoniopropylamine (DMSP-amine), and DMSP. The 35S-labeling kinetics of SMM and DMSP-amine indicated that they were intermediates and, consistent with this, the dimethylsulfonium moiety of SMM was shown by stable isotope labeling to be incorporated as a unit into DMSP. The identity of DMSP-amine, a novel natural product, was confirmed by both chemical and mass-spectral methods. S. alterniflora readily converted supplied [35S]SMM to DMSP-amine and DMSP, and also readily converted supplied [35S]DMSP-amine to DMSP; grasses that lack DMSP did neither. A small amount of label was detected in 3-dimethylsulfoniopropionaldehyde (DMSP-ald) when [35S]SMM or [35S]DMSP-amine was given. These results are consistent with the operation of the pathway Met → SMM → DMSP-amine → DMSP-ald → DMSP, which differs from that found in Compositae by the presence of a free DMSP-amine intermediate. This dissimilarity suggests that DMSP synthesis evolved independently in Gramineae and Compositae.

Osmotic Stress Induces Expression of Choline Monooxygenase in Sugar Beet and Amaranth1

Choline monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach (Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet (Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl2 = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mm salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family.

Salinity Promotes Accumulation of 3-Dimethylsulfoniopropionate and Its Precursor S-Methylmethionine in Chloroplasts1

Wollastonia biflora (L.) DC. plants accumulate the osmoprotectant 3-dimethylsulfoniopropionate (DMSP), particularly when salinized. DMSP is known to be synthesized in the chloroplast from S-methylmethionine (SMM) imported from the cytosol, but the sizes of the chloroplastic and extrachloroplastic pools of these compounds are unknown. We therefore determined DMSP and SMM in mesophyll protoplasts and chloroplasts. Salinization with 30% (v/v) artificial seawater increased protoplast DMSP levels from 4.6 to 6.0 μmol mg−1 chlorophyll (Chl), and chloroplast levels from 0.9 to 1.9 μmol mg−1 Chl. The latter are minimum values because intact chloroplasts leaked DMSP during isolation. Correcting for this leakage, it was estimated that in vivo about one-half of the DMSP is chloroplastic and that stromal DMSP concentrations in control and salinized plants are about 60 and 130 mm, respectively. Such concentrations would contribute significantly to chloroplast osmoregulation and could protect photosynthetic processes from stress injury. SMM levels were measured using a novel mass-spectrometric method. About 40% of the SMM was located in the chloroplast in unsalinized W. biflora plants, as was about 80% in salinized plants; the chloroplastic pool in both cases was approximately 0.1 μmol mg−1 Chl. In contrast, ≥85% of the SMM was extrachloroplastic in pea (Pisum sativum L.) and spinach (Spinacia oleracea L.), which lack DMSP. DMSP synthesis may be associated with enhanced accumulation of SMM in the chloroplast.