Genome-wide identification and characterization of microRNAs responding to ABA and GA in maize embryos during seed germination.

MicroRNAs (miRNAs) are an important class of non-coding small RNAs that regulate the expression of target genes through mRNA cleavage or translational inhibition. Previous studies have revealed their roles in regulating seed dormancy and germination in model plants such as Arabidopsis thaliana, rice (Oryza sativa) and maize (Zea mays). However, the miRNA response to exogenous gibberellic acid (GA) and abscisic acid (ABA) during seed germination in maize has yet to be explored. In this study, small RNA libraries were generated and sequenced from maize embryos treated with GA, ABA or double-distilled water as control. A total of 247 miRNAs (104 known and 143 novel) were identified, of which 45 known and 53 novel miRNAs were differentially expressed in embryos in the different treatment groups. In total, 74 (37 up-regulated and 37 down-regulated) and 55 (23 up-regulated and 32 down-regulated) miRNAs were expressed in response to GA and to ABA, respectively, and a total of 18 known and 38 novel miRNAs displayed differential expression between the GA- and ABA-treated groups. Using bioinformatics tools, we predicted the target genes of the differentially expressed miRNAs. Using GO enrichment and KEGG pathway analysis of these targets, we showed that miRNAs differentially expressed in our samples affect genes encoding proteins involved in the peroxisome, ribosome and plant hormonal signalling pathways. Our results indicate that miRNA-mediated gene expression influences the GA and ABA signalling pathways during seed germination.

TGFß1 protects myocardium from apoptosis and oxidative damage after ischemia reperfusion.

OBJECTIVE: Myocardial apoptosis is an important pathologic basis of ischemia-reperfusion injury (I/R). Transforming growth factor ß1 (TGFß1) participates in the regulation of oxidative damage and apoptosis. TGFß1 is upregulated in the repair process of I/R injury. It is speculated that TGFß1 over-expression is involved in the endogenous protective mechanism of I/R injury. This study explores the significance of TGFß1 in myocardial cell apoptosis after I/R. MATERIALS AND METHODS: Rat myocardial I/R injury model was established. Left ventricular ejection fraction (LVEF) and Left ventricular fractional shortening (LVFS) were detected by ultrasonic cardiogram. TGFß1 expression in the myocardium was tested. H9C2 cells were cultured under ischemic hypoxic condition for 6 h, and then were treated by reoxygenation for 6 h to simulate I/R model. H9C2 cells were divided into three groups, including I/R+pIRES2-Blank, I/R+pIRES2 TGFß1, and I/R+pIRES2-TGFß1+LY364947. TGFß1 mRNA and protein levels were evaluated. Cell apoptosis and reactive oxygen species (ROS) were determined by flow cytometry. RESULTS: LVEF and LVFS significantly decreased in I/R group compared with Sham group. TGFß1 mRNA and protein expressions in myocardium from I/R group up-regulated than the control. I/R treatment markedly elevated TGFß1 mRNA and protein levels, increased ROS content, and enhanced cell apoptosis in H9C2 cells. Over-expression of TGFß1 significantly weakened ROS production and apoptosis in H9C2 cells after I/R. TGFß receptor inhibitor LY364947 restrained ROS production and apoptosis attenuation in H9C2 cells treated by TGFß. CONCLUSIONS: TGFß1 alleviates myocardial cell apoptosis after I/R. Blocking TGFß1 attenuates the protective effect of TGFß1 on I/R injury.

Electronic Structure of Ni2E2 Complexes (E = S, Se, Te) and a Global Analysis of M2E2 Compounds: A Case for Quantized E2(n-) Oxidation Levels with n = 2, 3, or 4.

The diamagnetic compounds Cp'2Ni2E2 (1: E = S, 2: E = Se, 3: E = Te; Cp' = 1,2,3,4,-tetraisopropylcyclopentadienyl), first reported by Sitzmann and co-workers in 2001 [Sitzmann, H.; Saurenz, D.; Wolmershauser, G.; Klein, A.; Boese, R. Organometallics 2001, 20, 700], have unusual E···E distances, leading to ambiguities in how to best describe their electronic structure. Three limiting possibilities are considered: case A, in which the compounds contain singly bonded E2(2-) units; case B, in which a three-electron E∴E half-bond exists in a formal E2(3-) unit; case C, in which two E(2-) ions exist with no formal E-E bond. One-electron reduction of 1 and 2 yields the new compounds [Cp*2Co][Cp'2Ni2E2] (1red: E = S, 2red: E = Se; Cp* = 1,2,3,4,5-pentamethylcyclopentadieyl). Evidence from X-ray crystallography, X-ray absorption spectroscopy, and X-ray photoelectron spectroscopy suggest that reduction of 1 and 2 is Ni-centered. Density functional theory (DFT) and ab initio multireference methods (CASSCF) have been used to investigate the electronic structures of 1-3 and indicate covalent bonding of an E2(3-) ligand with a mixed-valent Ni2(II,III) species. Thus, reduction of 1 and 2 yields Ni2(II,II) species 1red and 2red that bear unchanged E2(3-) ligands. We provide strong computational and experimental evidence, including results from a large survey of data from the Cambridge Structural Database, indicating that M2E2 compounds occur in quantized E2 oxidation states of (2 × E(2-)), E2(3-), and E2(2-), rather than displaying a continuum of variable E-E bonding interactions.

An "intermediate spin" nickel hydride complex stemming from delocalized Ni2(µ-H)2 bonding.

The nickel hydride complex [Cp'Ni(µ-H)]2 (1, Cp' = 1,2,3,4-tetraisopropylcyclopentadienyl) is found to have a strikingly short Ni-Ni distance of 2.28638(3) Å. Variable temperature and field magnetic measurements indicate an unexpected triplet ground state for 1 with a large zero-field splitting of +90 K (63 cm(-1)). Electronic structure calculations (DFT and CASSCF/CASPT2) explain this ground state as arising from half occupation of two nearly degenerate Ni-Ni π* orbitals.

Covalent attachment of catalyst molecules to conductive diamond: CO2 reduction using "smart" electrodes.

We report here covalent attachment of a catalytically active cobalt complex onto boron-doped, p-type conductive diamond. Peripheral acetylene groups were appended on a cobalt porphyrin complex, and azide-alkyne cycloaddition was used for covalent linking to a diamond surface decorated with alkyl azides. The functionalized surface was characterized by X-ray photoelectron spectroscopy and Fourier transform IR spectroscopy, and the catalytic activity was characterized using cyclic voltammetry and FTIR. The catalyst-modified diamond surfaces were used as "smart" electrodes exhibiting good stability and electrocatalytic activity for electrochemical reduction of CO(2) to CO in acetonitrile solution.

An example of the refinement of positional disorder modeled as compositional disorder in [5-(2-formylphenyl)-10,15,20-triphenylporphyrinato]nickel(II).

The title compound, [Ni(C(45)H(28)N(4)O)], crystallizes in the space group I ̅42d and resides on a crystallographic fourfold rotoinversion axis with only a quarter of the complex in the asymmetric unit. The complex displays positional disorder as the one aldehyde group on the ligand can be located at four different positions. It was necessary to model this as compositional disorder to obtain a correct model and refinement. The practical approach to the refinement is explained.

X-ray absorption spectroscopic, crystallographic, theoretical (DFT) and chemical evidence for a chalcogen-chalcogen two-center/three-electron half bond in an unprecedented "subselenide" Se2(3-) ligand.

Doing things by halves: The dimeric compound (Cp'Ni)(2)(µ(2)-Se(2)) (Cp' = 1,2,3,4-tetraisopropylcyclopentadienyl), shown in the scheme, was investigated by using low temperature X-ray crystallography and X-ray absorption spectroscopy. The Se K-edge energy strongly indicates a Se physical oxidation state of -1.5, consistent with an unprecedented two-center/three-electron half-bonded Se(2)(3-) or "subselenide" ion.

Bone structure and turnover in type 2 diabetes mellitus.

SUMMARY: We compared skeletal parameters in type 2 diabetic (T2DM) and non-diabetic postmenopausal women. Bone structure by dual energy x-ray absorptiometry (DXA) and HR-pQCT was not different, although procollagen type 1 amino-terminal propeptide (P1NP) and osteocalcin levels were lower in T2DM. INTRODUCTION: T2DM is associated with increased fracture risk, but, paradoxically, with higher cross-sectional bone density (BMD) as measured by DXA. We sought explanations to this puzzle by investigating detailed structural and biochemical skeletal parameters in T2DM. METHODS: Cross-sectional comparison of 25 postmenopausal T2DM women and 25 matched controls using DXA, high-resolution peripheral quantitative computed tomography (HR-pQCT) and biochemical bone turnover markers. RESULTS: BMD by DXA did not differ between T2DM and controls. HR-pQCT assessment also did not differ, with the exception of cortical area at the tibia, which tended to be lower in the diabetics (difference of 12 ± 6 [mean ± SD] mm, p = 0.06). P1NP and osteocalcin levels were lower in T2DM as compared to controls (P1NP, 34.3 ± 16 vs. 57.3 ± 28 ng/ml; p = 0.005; osteocalcin, 4.5 ± 2 vs. 6.2 ± 2 nmol/L; p = 0.001). CONCLUSIONS: Postmenopausal women with T2DM had lower levels of bone formation markers as compared to controls. Aside from a possible decrease in cortical bone area at a weight-bearing site, bone structure was not altered in T2DM. Lower bone turnover may be a skeletal parameter that is present in T2DM.

Vitamin D deficiency in HIV-infected postmenopausal Hispanic and African-American women.

UNLABELLED: We evaluated vitamin D status in HIV+ and HIV- postmenopausal African-American (AA) and Hispanic women. Most women (74-78%) had insufficient 25-hydroxyvitamin D (25OHD) levels, regardless of HIV status. 25OHD was lower in AA women and women lacking supplement use, providing support for screening and supplementation. Among HIV+ women, 25OHD was associated with current CD4 but not type of antiretroviral therapy. INTRODUCTION: To evaluate vitamin D status and factors associated with vitamin D deficiency and insufficiency in HIV-infected (HIV+) postmenopausal minority women. METHODS: In this cross-sectional study, 89 HIV+ and 95 HIV- postmenopausal women (33% AA and 67% Hispanic) underwent assessment of 25OHD, 1,25-dihydroxyvitamin D, parathyroid hormone, markers of bone turnover and bone mineral density by dual energy X-ray absorptiometry. RESULTS: The prevalence of low 25OHD did not differ by HIV status; the majority of both HIV+ and HIV- women (74-78%) had insufficient levels (<30 ng/ml). Regardless of HIV status, 25OHD was significantly lower in AA subjects, and higher in subjects who used both calcium and multivitamins. In HIV+ women on antiretroviral therapy (ART), 25OHD was directly associated with current CD4 count (r=0.32; p<0.01) independent of age, ethnicity, BMI, or history of AIDS-defining illness. No association was observed between 1,25(OH)(2)D and CD4 count or between serum 25OHD, 1,25(OH)(2)D or PTH and type of ART. CONCLUSIONS: In postmenopausal minority women, vitamin D deficiency was highly prevalent and associated with AA race and lack of supplement use, as well as lower current CD4 cell count. These results provide support for screening and repletion of vitamin D in HIV+ patients.

A nonsynonymous polymorphism in cannabinoid CB2 receptor gene is associated with eating disorders in humans and food intake is modified in mice by its ligands.

Marijuana use activates cannabinoid receptors (CB-Rs) producing several behavioral effects related to addiction, mood, and appetite. We investigated the association between CNR2 gene, which encodes cannabinoid CB2 receptor (CB2-R) and eating disorders in 204 subjects with eating disorders and 1876 healthy volunteers in Japanese population. The effect of treatment with CB2-R ligands on mouse food consumption was also determined. The CB2-R ligands used suppressed food intake in a time- and strain-dependent manner when food was available ad libitum and during the 12-h fast except, AM 630-the CB2-R antagonist that stimulated food consumption in food-deprived mice. There is an association between the R63Q polymorphism of the CNR2 gene and eating disorders (P = 0.04; Odds ratio 1.24, 95% CI, (1.01-1.53). These results suggest that cannabinoid CB2-R is involved in the endocannabinoid signaling mechanisms associated with the regulation of food intake and in eating disorders.

Mutations in IHH, encoding Indian hedgehog, cause brachydactyly type A-1.

Brachydactyly type A-1 (BDA-1; MIM 112500) is characterized by shortening or missing of the middle phalanges (Fig. 1a). It was first identified by Farabee in 1903 (ref. 2), is the first recorded example of a human anomaly with Mendelian autosomal-dominant inheritance and, as such, is cited in most genetic and biological textbooks. Here we show that mutations in IHH, which encodes Indian hedgehog, cause BDA-1. We have identified three heterozygous missense mutations in the region encoding the amino-terminal signaling domain in all affected members of three large, unrelated families. The three mutant amino acids, which are conserved across all vertebrates and invertebrates studied so far, are predicted to be adjacent on the surface of IHH.

Design of potent and selective human cathepsin K inhibitors that span the active site.

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.

Successive Ontario governments forced to grapple with problem of health care fraud.

Albert Shu of Willowdale, Ont., is the 1995 winner of the Amy Chouinard Memorial Essay Contest. Named in memory of long time CMAJ and Canadian Journal of Surgery contributor Amy Chouinard, the competition is intended to stimulate interest in medical and health-related writing among journalism students. The winning essay, written prior to Ontario's 1995 provincial election, examined health care fraud in the province and the impact of the photo health card that was introduced by the New Democrats party government.

Characterization of the metabolites of the peptidomimetic human immunodeficiency virus type 1 protease inhibitor SK&F 107461 in rats using liquid chromatography/mass spectrometry.

The metabolic fate of SK&F 107461 [Cbz-Ala-Ala-Phe psi [CHOHCH2] Gly-Val-Val-OMe], a potent and specific inhibitor of the protease encoded by human immunodeficiency virus type 1, in male Sprague-Dawley rats is described. SK&F 107461 is a hexapeptide analog containing a hydroxyethylene linkage in place of one of the peptide bonds, and in which the amino terminus is blocked with a carbobenzyloxy group and the carboxy terminus is modified to a methyl ester. The major metabolites of SK&F 107461 found in bile and urine after intravenous administration of 3H-labeled compound were characterized by LC/MS using either thermospray or continuous flow/FAB models of ionization. Approximately 80% of the administered radioactivity was recovered in the bile of bile duct-exteriorized rats following an intravenous dose. Radiochromatographic profiling indicated that SK&F 107461 was subject to extensive biotransformation. Structures were determined for three major biliary and five major urinary metabolites. Two of the major circulating plasma metabolites observed after intravenous bolus administration had similar retention times to metabolites that were observed in both bile and urine. A pathway for the biotransformation of SK&F 107461 in the rat is proposed. The parent molecule underwent two primary modes of metabolism. Hydrolysis of the carboxy-terminal ester or hydrolysis of the Ala-Ala peptide bond near the amino terminus were the primary metabolic events. All of the other metabolites characterized can be accounted for by exopeptidase activity subsequent to one or both of these primary events. There were no major metabolites observed resulting from anything other than hydrolysis of the ester or peptide bonds in the parent molecule.

Synthesis of a tritiated human growth hormone releasing peptide.

Tritium-labeled growth hormone releasing peptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 was synthesized by tritium-halogen exchange on the precursor His-5,7-Br2-D-Trp-Ala-Trp-D-Phe-Lys-NH2. The radiolabeled peptide had a specific activity of 29 Ci/mmol and a radiochemical purity of 95%. The tritium label was shown by 3H NMR to be located mostly at the expected 5,7-positions of the indole nucleus in the D-Trp residue. The dibromopeptide was prepared by solid-phase peptide synthesis, employing racemic 5,7-Br2-Trp as a building block and separation of the resulting epimeric mixture by HPLC. 5,7-Br2-Trp was prepared by a five-step sequence beginning with 2,4-dibromoaniline. The use of anisole as an additive in the HF resin/peptide cleavage was rejected because anisole was found to undergo electrophilic substitution of the dibromoindole nucleus; a modified HF deprotection/cleavage procedure was developed and used instead.

Characterization of [3H]SK&F 108566 as a radioligand for angiotensin type-1 receptor.

Rat aortic smooth muscle cells were used as a model system to characterize the binding properties of [3H]SK&F 108566, an angiotensin type-1 (AT1) receptor antagonist. The binding was specific, saturable and reversible. The association and dissociation rates of [3H]SK&F 108566 binding to smooth muscle cells were monophasic and Scatchard analysis of equilibrium binding data yielded a linear plot indicating a homogenous population of binding sites. The maximum binding (Bmax) and apparent dissociation constant (Kd) were 22,000 +/- 6000 sites/cell and 0.83 +/- 0.08nM respectively. The pharmacological specificity of [3H]SK&F 108566 binding to smooth muscle cells is consistent with that observed for AT1 and confirms AT1 receptor specificity of this radioligand. High affinity binding was observed in membranes prepared from bovine adrenal cortex, rat liver and rat kidney glomeruli. COS cells transfected with cDNA encoding human AT1 angiotensin II receptors also displayed high affinity binding site for [3H]SK&F 108566. No specific binding could be detected on membranes prepared from bovine cerebellum, a tissue rich in the angiotensin type-2 (AT2) receptor. These observations indicate that [3H]SK&F 108566 binds to sites which have pharmacological characteristics of angiotensin II AT1 subtype receptors and can be used as a subtype-selective radioligand to characterize AII receptors in various systems.

A radiometric assay for HIV-1 protease.

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

Binding of a growth hormone releasing hexapeptide to specific hypothalamic and pituitary binding sites.

The drug SK&F 110679 (His-D-Trp-Ala-Trp-D-Phe-LysNH2), is an enkephalin-derived hexapeptide, which specifically releases growth hormone in a wide variety of species in vivo and in vitro. Previous binding studies, using ligands which are specific for mu and delta opioid binding sites, demonstrated an inverse relationship between the opioid binding potency and the potency in releasing growth hormone of a series of peptides related to SK&F 110679. In an attempt to understand its mode of action better, a binding assay for the peptide was established using a ligand which had been tritium labelled at the D-Trp2 residue. Membrane fragments from both the hypothalamus and anterior pituitary tissue were found to contain sites to which [3H]SK&F 110679 reversibly and saturably bound. The binding curves for [3H]SK&F 110679 to membrane fragments of both hypothalamus and anterior pituitary were resolved into two binding components with the computer program LIGAND. The Kd's obtained were in the 10(-8) M and 10(-5) M range. The relationship of these binding sites to the growth hormone-releasing activity of the peptide was explored by examining the relationship between the binding and potency in releasing growth hormone of a series of peptides related to SK&F 110679. For sites in both the hypothalamus and pituitary, a significant correlation between binding and the release of growth hormone was obtained. Thus, these binding sites appeared to be involved in the release of growth hormone by SK&F 110679-related peptides.(ABSTRACT TRUNCATED AT 250 WORDS)