Post annealing effect on the electrical properties of top-gate SWNT network transistors.

High performance top-gate single walled carbon nanotube network transistors are fabricated with aluminum oxide (Al2O3) layer as a gate dielectric by atomic layer deposition. It exhibits large on/off ratio (>10(4)) due to selective growth of semiconducting tubes by the plasma enhanced chemical vapor deposition. I-V characteristics show p-type or n-type depending on the deposition temperature. We investigate the type dependent characteristics for the carrier polarities with the post annealing effect on the top-gate SWNT network transistors. The dramatic change in the polarity of the top-gate SWNT network transistors, from n-type to p-type due to conversion of I-V characteristics is observed by post-annealing at 350 degrees C for 30 minutes under vacuum. Our observation suggests that competition between electron transfer from the Al2O3 layers to the SWNT surface and electron capture by oxygen molecules adsorbed on the tube walls seems to be the key point for the V(th) change as a function of Al2O3 deposition temperature.

Beta-secretase (BACE1)-inhibiting stilbenoids from Smilax Rhizoma.

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of Smilax Rhizoma (the dried rhizomes of Smilax china L.) showed potent inhibitory activity. The active compounds were identified as a trans/cis-resveratrol mixture, oxyresveratrol, veraphenol, and cis-scirpusin A. They were shown to non-competitively inhibit BACE1 with the Ki values of 5.4 x 10(-6), 5.4 x 10(-6), 3.4 x 10(-6), and 5.4 x 10(-6)M and IC(50) values of 1.5 x 10(-5), 7.6 x 10(-6), 4.2 x 10(-6), and 1.0 x 10(-5)M, respectively. The active compounds were less inhibitory to alpha-secretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase, suggesting that they were relatively specific inhibitors of BACE1.

Production of a new wheat line possessing the 1BL.1RS wheat-rye translocation derived from Korean rye cultivar Paldanghomil.

The 1BL.1RS translocations between wheat ( Triticum aestivum L.) and rye ( Secale cereale L.) are widely used in bread wheat breeding programs, but all modern wheat cultivars with the 1BL.1RS have shown genetic vulnerability due to one rye source - a German cultivar, Petkus. We have developed, a new 1BL.1RS wheat-rye translocation line from the backcross of the F(1) hybrid of wheat cv. Olmil and rye cv. Paldanghomil, both cultivars from Korea. The GISH technique was applied to identify the presence of rye chromatin in 467 BC(1)F(6) lines selected from 77 BC(1)F(5) lines. Only one line, Yw62-11, showed wheat-rye translocated chromosomes, with a somatic chromosome number of 2n=42. C-banding patterns revealed that the translocated chromosome was 1BL.1RS, showing prominent bands in the terminal and sub-terminal regions of the short arm as well as in the centromeric region and terminal region of the long arm. This new 1BL.1RS translocation line formed 21 bivalents like common wheat at meiotic metaphase I, thereby showing complete homology.

Demineralized bone matrix, bone morphogenetic proteins, and animal models of spine fusion: an overview.

Preclinical investigations on the use of bone morphogenetic proteins (BMP) in the spine have yielded promising results. This has led to the preliminary introduction of these growth factors in controlled clinical trials. Initial data made available suggest that these differentiating factors will play a major role in the treatment of spinal disorders in the future. This article reviews key preclinical studies and their results that formed the basis for introduction into clinical trials. Non-primate and non-human primate models of spine fusion with BMP are reviewed objectively, and important issues regarding carrier, dose, and site of implantation are discussed. Finally, exciting new gene therapy research is discussed, with comments made on its applicability for the future.

Nonaccidental pediatric head injury: diffusion-weighted imaging findings.

OBJECTIVE: Diffusion-weighted imaging (DWI) reveals nonhemorrhagic posttraumatic infarction hours to days before conventional computed tomographic scanning or magnetic resonance imaging (MRI). We evaluated the diagnostic utility of DWI in children with nonaccidental head trauma. METHODS: The medical records and imaging examinations obtained between January 1998 and May 2000 for all children less than 2 years of age with presumed or suspected nonaccidental head injury were reviewed retrospectively. Twenty children who had undergone DWI within 5 days of presentation were included in the study. Computed tomographic scans, conventional MRI sequences, and DWI combined with apparent diffusion coefficient (ADC) maps were evaluated. RESULTS: Eleven girls and nine boys (median age, 5 mo) were studied. Eighteen children had presumed nonaccidental head trauma, and two children had suspected nonaccidental head trauma. Of the 18 children with presumed nonaccidental trauma, 16 (89%) demonstrated abnormalities on DWI/ADC, as compared with neither of the two children with suspected nonaccidental trauma. In 13 (81%) of 16 positive cases, DWI revealed more extensive brain injury than was demonstrated on conventional MRI sequences or showed injuries not observed on conventional MRI. DWI combined with ADC maps allowed better delineation of the extent of white matter injury. DWI/ADC abnormalities in the nonaccidental head-injured children were likely to involve posterior aspects of the cerebral hemispheres, with relative sparing of the frontal and temporal poles. Severity on DWI correlated significantly with poor outcome (P < 0.005). CONCLUSION: DWI has broad applications in the early detection of infarction in children with nonaccidental head injury and enhances the sensitivity of conventional MRI. In the patients in this study, early DWI provided an indicator of severity that was more complete than any other imaging modality. The use of DWI may help to identify children at high risk for poor outcome and to guide management decisions.

Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis.

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene-producing plants, Scoparia dulcis and Croton sublyratus, have been isolated using the homology-based polymerase chain reaction (PCR) method. Both clones contained highly conserved aspartate-rich motifs (DDXX(XX)D) and their N-terminal residues exhibited the characteristics of chloroplast targeting sequence. When expressed in Escherichia coli, both the full-length and truncated proteins in which the putative targeting sequence was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to produce geranylgeranyl diphosphate (GGPP). The structural factors determining the product length in plant GGPPSs were investigated by constructing S. dulcis GGPPS mutants on the basis of sequence comparison with the first aspartate-rich motif (FARM) of plant farnesyl diphosphate synthase. The result indicated that in plant GGPPSs small amino acids, Met and Ser, at the fourth and fifth positions before FARM and Pro and Cys insertion in FARM play essential roles in determination of product length. Further, when a chimeric gene comprised of the putative transit peptide of the S. dulcis GGPPS gene and a green fluorescent protein was introduced into Arabidopsis leaves by particle gun bombardment, the chimeric protein was localized in chloroplasts, indicating that the cloned S. dulcis GGPPS is a chloroplast protein.

Diverse chalcone synthase superfamily enzymes from the most primitive vascular plant, Psilotum nudum.

Psilotum nudum Griseb is a pteridophyte and belongs to the single family (Psilotaceae) of the division, Psilophyta. Being the only living species of a once populated division, P. nudum is the most primitive vascular plant. Chalcone synthase (CHS; EC 2.3.1.74) superfamily enzymes are responsible for biosyntheses of diverse secondary metabolites, including flavonoids and stilbenes. Using a reverse transcription-polymerase chain reaction strategy, four CHS-superfamily enzymes (PnJ, PnI, PnL and PnP) were cloned from P. nudum, and heterologously expressed in Escherichia coli. These four enzymes of 396-406 amino acids showed sequence identity of > 50% among themselves and to other higher-plant CHS-superfamily enzymes. PnJ and PnP preferred p-coumaroyl-CoA and isovaleryl-CoA respectively, as starter CoA and catalyzed CHS-type ring formation, indicating that they are CHS and phlorisovalerophenone synthase, respectively. On the other hand, PnI and PnL preferred cinnamoyl-CoA as starter CoA and catalyzed stilbene synthase-type cyclization and thus were determined to be pinosylvin synthases (EC 2.3.1.146). In addition, PnE, which uniquely contains a glutamine in place of otherwise strictly conserved histidine, had no apparent in vitro catalytic activity. Phylogenetic analysis indicated that these P. nudum clones form a separate cluster together with Equisetum arvense CHS. This cluster of pteridophytes is located next to the cluster formed by pine (gymnosperm) enzymes, in agreement with their evolutionary relationships. Psilotum nudum represents a plant with the most diverse CHS-superfamily enzymes and this ability to diverge may have provided a survival edge during evolution.

Pediatric supratentorial intraventricular tumors.

A variety of mass lesions can arise within or in proximity to the ventricular system in children. These lesions are relatively uncommon, and they present a unique diagnostic and surgical challenge. The differential diagnosis is determined by tumor location in the ventricular system, clinical presentation, age of the patient, and the imaging characteristics of the lesion. In this report the authors provide an introduction to and an overview of the most common pediatric supratentorial intraventricular tumors. The typical radiographic features of each tumor and location preference within the ventricular system are reviewed. Management and treatment considerations are discussed. Examination of tissue samples to obtain diagnosis is usually required for accurate treatment planning, and resection without adjuvant therapies is often curative. The critical management decision frequently involves determining which lesions are appropriate for surgical therapy. Careful preoperative neuroimaging is extremely useful in planning surgery. Knowledge of the typical imaging characteristics of these tumors can help to determine the diagnosis with relative certainty when a tissue sample has not been obtained, because a small subset of these lesions can be managed expectantly.

Lactate elicits vascular endothelial growth factor from macrophages: a possible alternative to hypoxia.

Macrophages respond to various stimuli to produce angiogenic factors but few mechanistic details are known. We examined the effects of hypoxia, lactate and nicotinamide on the expression of vascular endothelial growth factor by cultured macrophages. These agents were chosen because they down-regulate polyadenosine diphosphoribose levels. Following exposure, conditioned media were analyzed for vascular endothelial growth factor protein. Nicotinamide adenine dinucleotide, polyadenosine diphosphoribose, and vascular endothelial growth factor mRNA were measured in the cellular fraction. Angiogenic capacity of the conditioned media was tested in rabbit corneas and Matrigel implants. All three agents, hypoxia, lactate and nicotinamide, elicited significantly increased levels of vascular endothelial growth factor mRNA and vascular endothelial growth factor in the conditioned media, and these levels were paralleled by their angiogenic activity. Polyadenosine diphosphoribose in the cellular fraction was correspondingly depressed. Anti-vascular endothelial growth factor antibody inhibited most of the angiogenic response whereas anti-basic fibroblast growth factor antibody had little effect. We propose that redox changes associated with the alteration of cellular nicotinamide adenine dinucleotide and polyadenosine diphosphoribose are involved in lactate-mediated VEGF expression.

Evidence for catalytic cysteine-histidine dyad in chalcone synthase.

Chalcone and stilbene synthases (CHS and STS) catalyze condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA, but catalyze different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Condensing activities of wild-type CHS and STS as well as STS-C60S mutant were inhibited by iodoacetamide (Idm) and diethyl pyrophosphate (DPC). DPC also inhibited malonyl-CoA decarboxylation activity of wild-type and C164S mutants of CHS and STS. Meanwhile, Idm treatment enhanced (two- to fourfold) malonyl decarboxylase activity of wild-type enzymes and STS-C60S, whereas this priming effect was not observed with C164S mutants of CHS and STS, indicating that the cysteine residue being modified by Idm is the catalytic Cys164 of CHS and STS. DPC inhibition of decarboxylation activity of wild-type CHS was pH-independent in the range of pH 5.8 to 7.8; however, its inhibitory effect on CHS-C164S increased as pH increased from 6.2 to 7.4 with a midpoint of 6.4. Based on the 3-D structure of CHS and the observed shift in microscopic pK(a), it was concluded that the histidine residue being modified by DPC in CHS is likely the catalytic His303 and that His303 forms an ionic pair (catalytic dyad) with Cys164 in wild-type CHS. In addition, our results showed that Cys60 in STS is not essential for the activity and only a single cysteine (Cys164) participates in the catalysis as in CHS.

Identification of amino acid residues important in the cyclization reactions of chalcone and stilbene synthases.

Chalcone synthase (CHS) and stilbene synthase (STS) catalyse condensation reactions of p-coumaroyl-CoA and three C(2) units from malonyl-CoA up to a common tetraketide intermediate but then catalyse different cyclization reactions to produce naringenin chalcone and resveratrol respectively. On the basis of sequence alignment with other condensing enzymes including 3-ketoacyl-(acyl carrier protein) synthases of polyketide and fatty-acid synthases, site-directed mutagenesis was performed on the active-site G(372)FGPG loops in CHS and STS. The CHS-P375G mutant showed a 6-fold decrease in overall condensing activity with selectively increased production of p-coumaroyltriacetic acid lactone (CTAL, the derailment product of the tetraketide intermediate). Meanwhile, resveratrol production by STS-P(375)G strongly decreased to give various products in the order CTAL> resveratrol approximately bisnoryangonin>naringenin. As a result, naringenin production (cross-reaction) by STS-P(375)G was close to 30% of resveratrol production. Both G(374)L mutants of CHS and STS showed no condensing activity with residual malonyl-CoA decarboxylase activity. These results suggested that the G(372)FGPG loop in CHS and STS contribute to a determination of the outcome during cyclization reactions by serving as a part of the active-site scaffold on which the stereochemistry of cyclization is performed. These observations provide the first biochemical indication that cyclization reactions are modulated by active-site geometry. The implications for the evolutionary relationship of these enzymes are also discussed.

Chalcone and stilbene synthases expressed in eucaryotes exhibit reduced cross-reactivity in vitro.

Chalcone synthase (CHS) and stilbene synthase (STS) catalyze different cyclization reactions of the common tetraketide to give different products, naringenin chalcone and resveratrol, respectively. We have previously observed in vitro cross-reaction of CHS and STS overexpressed in Escherichia coli, resveratrol production by CHS and chalcone production by STS. When expressed in eucaryotic cells, or in E. coli as thioredoxin-fusion proteins, CHS and STS exhibited reduced cross-reaction. STS refolded from inclusion bodies also showed reduced cross-reaction. While addition of bovine serum albumin and pH in the reaction were without noticeable effect, addition of glycerol decreased the cross-reaction of CHS likely due to its stabilizing effect on enzyme conformation. These results were interpreted to provide supporting evidence to our earlier proposition (Yamaguchi T. et al., FEBS Lett., 460, 457-4 61 (1999)) that the in vitro cross-reaction of CHS and STS is due to intrinsic capability of these enzymes to catalyze different types of cyclization, which, in turn, is endowed by conformational flexibility of their active sites.

Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus. cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase.

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated using the homology-based polymerase chain reaction method. Both cloned genes showed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs. The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS activity to catalyze the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate to form geranylgeranyl diphosphate. To investigate the factor determining the product chain length of plant GGPPSs, S. dulcis GGPPS mutants in which either the small amino acids at the fourth and fifth positions before the first aspartate-rich motif (FARM) were replaced with aromatic amino acids or in which two additional amino acids in FARM were deleted were constructed. Both mutants behaved like FPPS-like enzymes and almost exclusively produced FPP when dimethylallyl diphosphate was used as a primer substrate, and failed to accept FPP as a primer substrate. These results indicate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product length determination in plant GGPPSs.

Understanding angiogenesis and its clinical applications.

Angiogenesis is the growth of new vessels from pre-existing blood vessels. Angiogenesis is critical during embryogenesis but occurs minimally in healthy adults, except in wound repair, inflammation, female reproductive organs, and pathologic conditions. Various growth factors and proteins, elements of the extracellular matrix, components of the coagulation/fibrinolytic system, and platelets interact with the endothelial cells and pericytes of blood vessels to regulate angiogenesis. Characterization of angiogenic factors has revealed that remodeling of the extracellular matrix occurs during angiogenesis, mediated by integrins that are found on the endothelial cell surface membrane. Counter-regulatory antiangiogenic proteins and molecules that show an intricate balance in the regulation of angiogenesis have also been characterized. Components of the coagulation/fibrinolysis cascade also play a critical role in angiogenesis. Elucidation of the mechanisms of angiogenesis has led to better understanding of certain disease states. Ongoing studies are evaluating the stimulation of angiogenesis to treat ischemic disorders, and the inhibition of angiogenesis to prevent abnormal proliferation in malignant and non-malignant disorders.

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli.

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7-4.2% of naringenin) and naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.

Different inhibitory effects of 5-S-glutathionyl-beta-alanyl-L-dopa (5-S-GA-L-D) analogues on autophosphorylation and substrate phosphorylation of Src protein tyrosine kinase.

Starting with 5-S-glutathionyl-beta-alanyl-L-dopa (1) and 5-S-glutathionyl-beta-alanyl-dopamine (2), a series of analogues with truncated glutathionyl and beta-alanyl-dopa moieties were synthesized, and their inhibitory effects on autophosphorylation and substrate phosphorylation reaction by c-Src and by epidermal growth factor receptor (EGFR) were evaluated. When the glutamyl residue was removed, the inhibitory effects on v-Src autophosphorylation decreased about 4- to 5-fold, and concomitant removal of the glutamyl and beta-alanyl residues resulted in a 40- to 60-fold decrease in the inhibition of v-Src autophosphorylation. On the other hand, these modifications had little effect on the inhibitory activity of substrate (Raytide) phosphorylation by c-Src. Interestingly, 5-S-cysteinyl dopamine inhibited the Src substrate phosphorylation reaction with comparable potency to that of genistein. Nonpeptide lipophilic derivatives had a similar inhibition on v-Src autophosphorylation but decreased inhibitory effects on substrate phosphorylation when compared to the lead compounds. Most compounds showed little effect on substrate phosphorylation by EGFR.

In vitro platelet-activating factor receptor binding inhibitory activity of pinusolide derivatives: a structure-activity study.

Pinusolide, a labdane-type diterpene lactone isolated from Biota orientalis, was found to be a potent platelet-activating factor (PAF) receptor binding antagonist. To investigate the structure-activity relationship and find derivatives with improved pharmacological profiles, 17 pinusolide derivatives were prepared and tested for their ability to inhibit the PAF receptor binding. The results demonstrated that the carboxymethyl ester group at C-19, the integrity of the alpha,beta-unsaturated butenolide ring, and the exocyclic olefinic function of pinusolide are all necessary for its maximum PAF receptor binding inhibitory activity. Among the derivatives, the 17-nor-8-oxo derivative 8 was found to be as potent as pinusolide. The results also suggested that several derivatives warrant further pharmaceutical and pharmacological studies due to their improved water solubility (8 and 11) and apparent lack of susceptibility to Michael-type nucleophilic addition (13 and 18).

Time line of wound healing.

The time line of wound healing is altered by various local conditions, such as inflammation and neuropathy; however, the most important factor regulating the regional time line of healing is blood flow. Factors that can increase oxygen delivery to the regional tissue, such as supplemental oxygen, warmth, and sympathetic blockade, can accelerate the time line of healing.

Mice lacking the thrombin receptor, PAR1, have normal skin wound healing.

Thrombin's actions on platelets, macrophages, fibroblasts, and endothelial cells have prompted the hypothesis that thrombin may be important for inflammatory and fibroproliferative processes in wound healing. Protease-activated receptor 1 (PAR1) is a G-protein-coupled receptor that mediates many of the cellular activities of thrombin. To test the role of this receptor in vivo, we generated PAR1-deficient mice. Despite the observation that fibroblasts cultured from these mice lacked responsiveness to thrombin in vitro, we now report that there was no difference detected between wild-type and PAR1-deficient mice in skin wound healing assays including time to closure of open wounds, tensile strength of healed incisional wounds, wound histology, and hydroxyproline/DNA content of wound implants. We conclude that PAR1 is not necessary for normal skin wound healing in mice.

Corpus callosal changes associated with hydrocephalus: a report of two cases.

OBJECTIVE AND IMPORTANCE: Focal or diffuse corpus callosal changes can occur in patients with active hydrocephalus who undergo shunting procedures. The neural compression caused by active hydrocephalus and the conditions that follow ventricular shunting may contribute to the development of these changes. CLINICAL PRESENTATION: Two patients who underwent successful shunting for hydrocephalus subsequently developed thickening and diffuse signal changes in the corpus callosum, which were revealed by magnetic resonance imaging. The abnormal signal intensity extended laterally and linearly along the callosal fiber tracts and was not associated with mass effect. These changes persisted despite clinical improvement after the shunts were implanted. INTERVENTION: Detailed neuropsychological testing showed no evidence of residual cognitive impairment or any interruption of the interhemispheric transfer of information. It has been proposed that the impingement of the corpus callosum by the rigid falx may contribute to symptomatic hydrocephalus. Impingement may cause partial hemispheric disconnection, resulting from callosal axonal dysfunction. Our patients showed radiographic evidence of dramatic changes within the corpus callosum after ventricular shunting, consistent with a transcallosal demyelinating process. Patients demonstrated neither clinical nor neuropsychological evidence of callosal disconnection, even though the callosal changes persisted. In these two patients, it is reasonable to assume that the relative sparing of the splenium accounts for the lack of neuropsychological deficits. CONCLUSION: Based on our findings, conservative management, rather than a stereotactic biopsy or other forms of intervention, seems reasonable when these characteristic changes of the callosum are noted by magnetic resonance imaging after a shunt for hydrocephalus has been implanted in the patient.