The evolutionary fate of the chloroplast and nuclear rps16 genes as revealed through the sequencing and comparative analyses of four novel legume chloroplast genomes from Lupinus.

The Fabaceae family is considered as a model system for understanding chloroplast genome evolution due to the presence of extensive structural rearrangements, gene losses and localized hypermutable regions. Here, we provide sequences of four chloroplast genomes from the Lupinus genus, belonging to the underinvestigated Genistoid clade. Notably, we found in Lupinus species the functional loss of the essential rps16 gene, which was most likely replaced by the nuclear rps16 gene that encodes chloroplast and mitochondrion targeted RPS16 proteins. To study the evolutionary fate of the rps16 gene, we explored all available plant chloroplast, mitochondrial and nuclear genomes. Whereas no plant mitochondrial genomes carry an rps16 gene, many plants still have a functional nuclear and chloroplast rps16 gene. Ka/Ks ratios revealed that both chloroplast and nuclear rps16 copies were under purifying selection. However, due to the dual targeting of the nuclear rps16 gene product and the absence of a mitochondrial copy, the chloroplast gene may be lost. We also performed comparative analyses of lupine plastomes (SNPs, indels and repeat elements), identified the most variable regions and examined their phylogenetic utility. The markers identified here will help to reveal the evolutionary history of lupines, Genistoids and closely related clades.

The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): Comparative analyses and molecular dating.

The history of many plant lineages is complicated by reticulate evolution with cases of hybridization often followed by genome duplication (allopolyploidy). In such a context, the inference of phylogenetic relationships and biogeographic scenarios based on molecular data is easier using haploid markers like chloroplast genome sequences. Hybridization and polyploidization occurred recurrently in the genus Spartina (Poaceae, Chloridoideae), as illustrated by the recent formation of the invasive allododecaploid S. anglica during the 19th century in Europe. Until now, only a few plastid markers were available to explore the history of this genus and their low variability limited the resolution of species relationships. We sequenced the complete chloroplast genome (plastome) of S. maritima, the native European parent of S. anglica, and compared it to the plastomes of other Poaceae. Our analysis revealed the presence of fast-evolving regions of potential taxonomic, phylogeographic and phylogenetic utility at various levels within the Poaceae family. Using secondary calibrations, we show that the tetraploid and hexaploid lineages of Spartina diverged 6-10 my ago, and that the two parents of the invasive allopolyploid S. anglica separated 2-4 my ago via long distance dispersal of the ancestor of S. maritima over the Atlantic Ocean. Finally, we discuss the meaning of divergence times between chloroplast genomes in the context of reticulate evolution.

The impact of LR-HSQMBC very long-range heteronuclear correlation data on computer-assisted structure elucidation.

The impact of LR-HSQMBC very long-range (n)JCH heteronuclear shift correlation data as a supplement to HMBC data as input for the computer-assisted structure elucidation program, Structure Elucidator(®), is assessed for the first time. The severely proton-deficient xanthone antibiotic cervinomycin A2 and the alkaloid staurosporine were employed as a model compounds.

Consistency between research and clinical diagnoses of autism among boys and girls with fragile X syndrome.

BACKGROUND: Prior research suggests that 60-74% of males and 16-45% of females with fragile X syndrome (FXS) meet criteria for autism spectrum disorder (ASD) in research settings. However, relatively little is known about the rates of clinical diagnoses in FXS and whether such diagnoses are consistent with those performed in a research setting using gold standard diagnostic tools. METHOD: This study explored whether boys and girls with FXS met criteria for ASD in a research setting using the Autism Diagnostic Observation Schedule (ADOS) and the Autism Diagnostic Interview-Revised (ADI-R), and then compared these data with the frequency of parent-reported clinical diagnoses. We also examined child and family characteristics as potential diagnostic predictors across settings. Participants included 35 females and 51 males with FXS (mean age: 10 years), who were from Eastern and Midwestern regions of the USA. RESULTS: About half of the children met criteria for ASD on either the ADOS or ADI-R, with ASD occurring three times more frequently in males than females (∼75% vs. ∼25%). In contrast, ∼25% of participants of both genders had received a clinical diagnosis of ASD. While cognitive and language skills predicted diagnostic outcome on the ADOS and ADI-R, these skills did not predict clinical diagnoses. Executive functions predicted clinical diagnoses, but not diagnoses per the ADOS or ADI-R. CONCLUSIONS: ASD in FXS may be under-diagnosed in clinical/educational settings, which raises questions regarding access to ASD-related services.

Therapy service use among individuals with fragile X syndrome: findings from a US parent survey.

BACKGROUND: Fragile X syndrome (FXS) is known to be associated with a range of developmental challenges, yet the occurrence and intensity of therapy services along with associated factors have not been determined. METHOD: In a US national survey, caregivers provided information regarding the therapy services received by their sons (n = 1013) and daughters (n = 283) with FXS (from birth to 63 years; mean = 15.6 years, SD = 10.6). Caregivers reported (1) type, (2) amount, (3) location, and (4) overall satisfaction with services. Associations with other child variables and family income were also examined. RESULTS: Key findings included that 72% of males and 47% of females were currently receiving at least one type of therapy service; the most common services for both males and females were speech-language therapy (ST) and occupational therapy (OT). Overall, males were more likely to receive therapy services as well as a greater number of services than females. Autism status was significantly associated with both males and females receiving ST and males receiving OT and behaviour management therapy. Therapies were provided in a variety of locations, and parents were generally satisfied with the amount and quality of therapy services. Age-related declines were evident in the use of services for both males and females, with very few individuals receiving any therapy services after 20 years of age. CONCLUSIONS: This study provides a baseline description of the current state of therapy services for children with FXS, laying a foundation for future research and recommendations for service provision and policy.

Compartmentalized beta subunit distribution determines characteristics and ethanol sensitivity of somatic, dendritic, and terminal large-conductance calcium-activated potassium channels in the rat central nervous system.

Neurons are highly differentiated and polarized cells, whose various functions depend upon the compartmentalization of ion channels. The rat hypothalamic-neurohypophysial system (HNS), in which cell bodies and dendrites reside in the hypothalamus, physically separated from their nerve terminals in the neurohypophysis, provides a particularly powerful preparation in which to study the distribution and regional properties of ion channel proteins. Using electrophysiological and immunohistochemical techniques, we characterized the large-conductance calcium-activated potassium (BK) channel in each of the three primary compartments (soma, dendrite, and terminal) of HNS neurons. We found that dendritic BK channels, in common with somatic channels but in contrast to nerve terminal channels, are insensitive to iberiotoxin. Furthermore, analysis of dendritic BK channel gating kinetics indicates that they, like somatic channels, have fast activation kinetics, in contrast to the slow gating of terminal channels. Dendritic and somatic channels are also more sensitive to calcium and have a greater conductance than terminal channels. Finally, although terminal BK channels are highly potentiated by ethanol, somatic and dendritic channels are insensitive to the drug. The biophysical and pharmacological properties of somatic and dendritic versus nerve terminal channels are consistent with the characteristics of exogenously expressed alphabeta1 versus alphabeta4 channels, respectively. Therefore, one possible explanation for our findings is a selective distribution of auxiliary beta1 subunits to the somatic and dendritic compartments and beta4 to the terminal compartment. This hypothesis is supported immunohistochemically by the appearance of distinct punctate beta1 or beta4 channel clusters in the membrane of somatic and dendritic or nerve terminal compartments, respectively.

Applying computer-assisted structure elucidation algorithms for the purpose of structure validation: revisiting the NMR assignments of hexacyclinol.

Computer-assisted structure elucidation (CASE) using a combination of 1D and 2D NMR data has been available for a number of years. These algorithms can be considered as "logic machines" capable of deriving all plausible structures from a set of structural constraints or "axioms", defined by the spectroscopic data and associated chemical information or prior knowledge. CASE programs allow the spectroscopist not only to determine structures from spectroscopic data but also to study the dependence of the proposed structure on changes to the set of axioms. In this article, we describe the application of the ACD/Structure Elucidator expert system to help resolve the conflict between two different hypothetical hexacyclinol structures derived by different researchers from the NMR spectra of this complex natural product. It has been shown that the combination of algorithms for both structure elucidation and structure validation delivered by the expert system enables the identification of the most probable structure as well as the associated chemical shift assignments.

Controlling nitrate leaching in irrigated agriculture.

The impact of improved irrigation and nutrient practices on ground water quality was assessed at the Nebraska Management System Evaluation Area using ground water quality data collected from 16 depths at 31 strategically located multilevel samplers three times annually from 1991 to 1996. The site was sectioned into four 13.4-ha management fields: (i) a conventional furrow-irrigated corn (Zea mays L.) field; (ii) a surge-irrigated corn field, which received 60% less water and 31% less N fertilizer than the conventional field; (iii) a center pivot-irrigated corn field, which received 66% less water and 37% less N fertilizer than the conventional field; and (iv) a center pivot-irrigated alfalfa (Medicago sativa L.) field. Dating (3H/3He) indicated that the uppermost ground water was <1 to 2 yr old and that the aquifer water was stratified with the deepest water approximately 20 yr old. Recharge during the wet growing season in 1993 reduced the average NO3-N concentration in the top 3 m 20 mg L(-1), effectively diluting and replacing the NO3-contaminated water. Nitrate concentrations in the shallow zone of the aquifer increased with depth to water. Beneath the conventional and surge-irrigated fields, shallow ground water concentrations returned to the initial 30 mg NO3-N L(-1) level by fall 1995; however, beneath the center pivot-irrigated corn field, concentrations remained at approximately 13 mg NO3-N L(-1) until fall 1996. A combination of sprinkler irrigation and N fertigation significantly reduced N leaching with only minor reductions (6%) in crop yield.

Thalibealine, a novel tetrahydroprotoberberine--aporphine dimeric alkaloid from Thalictrum wangii.

A novel tetrahydroprotoberberine-aporphine dimeric alkaloid, (-)-thalibealine (1), was isolated from the roots of Thalictrum wangiii, and its structure established via spectroscopic analysis. Three other alkaloids were isolated, including the benzyltetrahydroisoquinoline-aporphine dimer (+)-thalmelatidine, the aporphine (+)-magnoflorine, and the protoberberine berberine. This is the first reported isolation of a tetrahydroprotoberberine-aporphine dimer from nature, as well as the first reported isolation of constituents from Thalictrum wangii.

Synthesis of thalprzewalskiinone, a revision of structure.

A direct comparison of the spectral data for synthetic 2-methyl-6,7-dimethoxy-3'-methoxy-4'-hydroxyoxobenzylisoquinoline iodide (1) and its positional isomer 2-methyl-6,7-dimethoxy-3'-hydroxy-4'-methoxyoxobenzylisoquinoline iodide (2) with the data obtained for the oxobenzylisoquinoline alkaloid thalprzewalskiinone revealed that the original structural assignment of the alkaloid as 1 was in error. These results mandate the revision of structure of thalprzewalskiinone to 2-methyl-6,7-dimethoxy-3'-hydroxy-4'-methoxyoxobenzylisoquinoline iodide (2).

NADPH:protochlorophyllide oxidoreductase from Synechocystis: overexpression, purification and preliminary characterisation.

NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. POR from the cyanobacterium Synechocystis has been overproduced in Escherichia coli with a hexahistidine tag at the N-terminus. This enzyme (His(6)-POR) has been purified to homogeneity and a preliminary characterisation of its kinetic and substrate binding properties is presented. Chemical modification experiments have been used to demonstrate inhibition of POR activity by the thiol-specific reagent N-ethyl maleimide. Substrate protection experiments reveal that the modified Cys residues are involved in either substrate binding or catalysis.

A comparison of inverse-detected heteronuclear NMR performance: conventional vs cryogenic microprobe performance.

We report a comparison of the results obtained at 500 MHz for heteronuclear shift correlation (HSQC) experiments with very small natural product samples using conventional and cryogenically cooled 3 mm NMR probes. The cryo probe affords a 12- to 16-fold reduction in data acquisition time for a comparable signal-to-noise ratio.

Long-range (1)H-(15)N heteronuclear shift correlation at natural abundance.

Despite the inherently low sensitivity of (15)N NMR because of its low gyromagnetic ratio (gamma(N)) and its relatively low natural abundance (0.37%), this important nuclide still has useful potential as a structural probe even at natural abundance. Inverse-detected NMR methods coupled with major advances in NMR probe designs have made it possible to acquire long-range (1)H-(15)N heteronuclear shift correlation data on samples as small as a micromole overnight. Chemical shift referencing schemes for (15)N and the range of (15)N shifts are discussed, followed by a discussion of the currently available pulse sequences, pulse calibration, parametrization and processing of long-range (1)H-(15)N data, and the implications of probe selection. These topics are followed by a review of the applications contained in the literature that have utilized (1)H-(15)N heteronuclear shift correlation experiments at natural abundance, with emphasis placed on the observed long-range coupling pathways.

Differential effects of the adenosine A(1) receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes.

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A(1) receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [3H]cyclohexyladenosine ([3H]CHA) to A(1) receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81, 723 (0.3-100 microM) produced a concentration-dependent enhancement of [3H]CHA binding to rat brain A(1) receptors. These effects were also species-dependent with larger enhancements (150-200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [3H]CHA binding to A(1) receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 microM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A(1) receptor selective antagonist 8-cyclopentyl-1, 3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 microM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [3H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 microM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A(1) receptor in a high-affinity state and that the relative proportions of high-affinity A(1) receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A(1) receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A(1) receptor.

Improved performance accordion heteronuclear multiple-bond correlation spectroscopy-IMPEACH-MBC.

A modification of the recently reported ACCORD-HMBC long-range heteronuclear shift correlation experiment is described. The new experiment, IMPEACH-MBC (improved performance accordion heteronuclear multiple-bond correlation), introduces a new pulse sequence element, a constant time variable delay. The incorporation of the constant time variable delay into the IMPEACH-MBC sequence suppresses (1)H-(1)H coupling modulation inherent to the utilization of the accordion principle to sample a broad range of potential long-range heteronuclear couplings. (1)H-(1)H coupling modulation, which introduces an F(1) modulation or a "skew" of responses in the second frequency domain of the ACCORD-HMBC experiment, is suppressed in the IMPEACH-MBC experiment. Results of identically optimized IMPEACH-MBC and ACCORD-HMBC experiments performed on a sample of strychnine are compared.

The relationship between clinical assessments of nutritional status and adverse outcomes in older hospitalized medical patients.

BACKGROUND: Malnutrition is common in hospitalized older people and may predict adverse outcomes. Previous studies of the relationship between nutritional status and hospital outcomes are limited by inadequate accounting for other potential predictors of adverse outcomes, the failure to consider functional outcomes, and the omission of clinical assessments of nutritional status. OBJECTIVE: To measure the relationship between a clinical assessment of nutritional status on hospital admission and subsequent mortality, functional dependence, and nursing home use. DESIGN: Prospective cohort study SETTING: A tertiary care hospital PATIENTS: A total of 369 patients at least 70 years old (mean age 80.3, 62% women) admitted to a general medical service MEASUREMENTS: Nutritional status was measured with the Subjective Global Assessment, a validated measure of nutritional status based on historical and physical exam findings. Patients were classified as severely malnourished (generally at least a 10% weight loss over the previous 6 months and marked physical signs of malnutrition), moderately malnourished (generally a 5 to 10% weight loss and moderate physical signs), or well nourished. Vital status, independence in activities of daily living, and nursing home use were determined through patient or surrogate interview at admission and 90 days and 1 year after discharge. Indices of comorbidity and illness severity were determined from chart review. RESULTS: 219 patients (59.3%) were well nourished, 90 (24.4%) were moderately malnourished, and 60 (16.3%) were severely malnourished. Severely malnourished patients were more likely than moderately malnourished or well nourished patients to die by 90 days (31.7%, 23.3%, and 12.3%, respectively, P < .001) and 1 year (55.0%, 35.6%, and 27.9%, P < .001) after discharge. In logistic regression models controlling for acute illness severity, comorbidity, and functional status on admission, severely malnourished patients were more likely than well nourished patients to die within 1 year of discharge (OR = 2.83, 95% CI, 1.47-5.45), to be dependent in activities of daily living 3 months after discharge (OR = 2.81, 1.06-7.46), and to spend time in a nursing home during the year after discharge (OR = 3.22, 1.05-9.87). CONCLUSION: Malnutrition was common in hospitalized patients with medical illness and was associated with greater mortality, delayed functional recovery, and higher rates of nursing home use. These adverse outcomes were not explained by greater acute illness severity, comorbidity, or functional dependence in malnourished patients on hospital admission.

Azepinoindole derivatives with high affinity for brain dopamine and serotonin receptors.

We synthesized 20 and 21 as conformationally constrained analogues of the dopamine receptor antagonist SKF-83742, as well as analogues 6-9, 16, and 18-22. Although 20 and 21 were inactive, 7, 9, and 19 showed strong binding to D-1, D-2, S-2, and alpha-1 receptors, as well as antipsychotic activity in vivo.

Galiposin: a new beta-hydroxychalcone from Galipea granulosa.

A novel beta-hydroxychalcone, galiposin, was isolated from the bark of Galipea granulosa. The structure of galiposin was established via spectroscopic analysis, including high resolution one- and two-dimensional nuclear magnetic resonance spectrometry, as well as mass spectroscopy.