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Aluminium nitride has good electrical, thermal, and mechanical properties, and Al4C3 is the most important intermediate in the preparation of AlN through carbothermal reduction. Ab initio molecular dynamics (AIMD) simulations were adopted in this study to elucidate the nitridation reaction mechanism of Al4C3, and an experimental study was conducted to verify the results of AIMD. Both the AIMD and experimental results showed that the nitridation pathway of Al4C3 was Al4C3→Al5C3N→AlN and that the nitridation of Al5C3N was considerably rapid. During the nitridation of Al4C3, the addition of carbon not only had a catalytic effect on the activation of nitrogen molecules but also promoted the aggregation of carbon atoms in the Al4C3 lattice to form elemental carbon.

Intramolecular amination of organoboronates occurs with a 1,2-metalate shift of an aminoboron "ate" complex to form azetidines, pyrrolidines, and piperidines. Bis(boronates) undergo site-selective amination to form boronate-containing azacycles. Enantiomerically enriched azacycles are formed with high stereospecificity.

In this paper is described an easily synthesized chiral diazaborolidine that is inexpensive, stable, and provides excellent stereoselection across a number of reaction classes. These versatile compounds possess utility in four different classes of cycloaddition reactions, offering good yield and stereoselectivity. X-ray structure analysis provides insight about the origin of stereocontrol.

Objective: The present study aims to examine whether secondhand smoke exposure (SHSE) in university students can affect three indices of self-satisfaction related to depression as indiced by appearance, weight, and freedom of life choice. Participants: We collected data from 740 nonsmoking students in the summer of 2018, of which 57.84% were exposed to secondhand smoke. Methods: Depressive symptoms, SHSE, smoking status, weight satisfaction, appearance satisfaction, and freedom of life choice were self-reported via a questionnaire. Results: The generalized linear analyses revealed that SHSE was linked to lower scores of perceived freedom of life choice but not significantly associated with weight nor appearance satisfaction. The mediation analyses indicated that perceived freedom of life choice fully mediated the association between SHSE and depressive symptoms. Conclusions: These findings shed light on the importance of SHSE and its effects on mental health in university students. Preventive strategies should therefore locally target university campuses.

Multiphase electrical machines are advantageous for many industrial applications that require a high power rating, smooth torque, power/torque sharing capability, and fault-tolerant capability, compared with conventional single three-phase electrical machines. Consequently, a significant number of studies of multiphase machines has been published in recent years. This paper presents an overview of the recent advances in multiphase permanent magnet synchronous machines (PMSMs) and drive control techniques, with a focus on dual-three-phase PMSMs. It includes an extensive overview of the machine topologies, as well as their modelling methods, pulse-width-modulation techniques, field-oriented control, direct torque control, model predictive control, sensorless control, and fault-tolerant control, together with the newest control strategies for suppressing current harmonics and torque ripples, as well as carrier phase shift techniques, all with worked examples.

The weather is arguably one of the most common topics in daily conversation. However, it is also a well-known factor in psychiatric problems worldwide such as depression, where particularly elders and females are prone to the condition. Previous studies have mostly focused on the association between long-term climate/season change and depressive symptoms to test the belief that people feel depressed when the weather is bad. Yet, little is known about the relationship between intraday weather conditions and depressive symptoms. This study aimed to decompose this association and explore age and gender differences in depressive symptoms’ report under different intraday weather conditions. We analyzed data with a total sample size of 20,987 individuals aged 15–96 years obtained via the China Labor-force Dynamic Survey (CLDS) project in 2016. The surveyors filled in the intraday weather conditions (i.e., sunny, cloudy, overcast, light rain, moderate rain, and heavy rain) in the questionnaire with depression being self-reported using the Center for Epidemiologic Studies Depression (CES-D) Scale. In the survey, 17.3% [95% confidence interval (CI) = 16.8%, 17.8%] individuals were assessed to have depressive symptoms (CES-D scores >15). Overcast day was the peak weather condition for depressive symptoms, while gender and age differences of depressive symptoms were biggest in the weather condition of moderate rain. Multivariate logistic regression revealed that female and middle-old age individuals were more likely to report having depressive symptoms, and people under overcast day were more likely to report having depressive symptoms compared to under sunny day [odds ratio (OR) = 1.25, 95%CI = 1.11, 1.39]. This intraday weather conditions-depression link was significant in males and middle-old age individuals but not in females and youngsters.

The geometrical structure, mean square displacement and electronic properties of ZnnInn(n = 2–10) alloy clusters have been systematically studied by means of density functional theory. The stability of zinc-indium clusters has been discussed. Ab-initio molecular dynamics were performed to exhaust search the global minimum structures of the clusters on the level of GGA-PBE by DMol3 package, Then, the structures were optimized and further calculated the mean square displacement, HOMO-LUMO gap and NBO on the level of B3LYP/LANL2DZ by using Gaussian program. The results show that as the size increases, the structure tends to be spherical, In atoms and Zn atoms are dispersed in the system, The binding force of In-In bond and Zn-In bond is stronger than that of Zn-Zn bond. Combined with our calculated results, it is found that with the increase of cluster size, the non-metallicity of clusters decreases while the metallicity of clusters increases, and even clusters are more stable than odd clusters. The interaction between zinc and indium can be analyzed at the atomic level to provide ideas for the separation and recovery of indium from zinc and indium in practical experiments.

In this paper, a novel nonconventional angle displacement (i.e., 15°) between two sets of three-phase windings is proposed for dual three-phase permanent-magnet (PM) machines. First, all feasible angle displacements between the two three-phase winding sets for various slot/pole number combinations of dual three-phase machines are discussed in general. Then, an in-depth investigation is carried out on a 24-slot/10-pole dual three-phase PM machine as an example covering various electromagnetic performances. The investigation shows that under healthy and three-phase open-circuit conditions, the proposed 15° configuration has a comparable electromagnetic performance to the 30° configuration, and a better performance than the 0° counterpart. Furthermore, under three-phase short-circuit (SC) condition, the proposed 15° configuration has the lowest SC current, smallest braking torque, and the best PM demagnetization withstand capability. Finally, three prototype 24-slot/10-pole surface-mounted PM machines are built and tested to verify the theoretical analyses.

This study presents the parasitic effects on different carrier signal injection methods with alternative sensing signals for the sensorless control of permanent magnet synchronous machines (PMSMs). The different carrier injection methods are the rotating injection in the stationary reference frame with carrier current sensing and with zero-sequence carrier voltage sensing, the pulsating injection in the estimated synchronous reference frame with carrier current sensing, and pulsating injection in the anti-rotating reference frame with zero-sequence carrier voltage sensing, respectively. The parasitic effects, e.g. the magnetic saturation, inverter non-linearity, iron and PM losses, torque ripple etc. will be fully analysed and compared for these different injection methods. All the theoretical analyses are validated by either finite-element analyses or experiments on a laboratory PM machine.

In this paper, the investigation is focused on the iron loss prediction for electrical machines fed by low switching frequency inverter. An improved iron loss model is developed after reviewing various existing iron loss models considering pulse-width modulation (PWM) influence. In order to validate the improved iron loss model, iron loss tests are carried out on an electrical machine. The predicted iron loss results match well with the measured results, which shows that the improved iron loss model is able to predict iron loss in electrical machine fed by an inverter. The comparison between the predicted iron losses with and without considering the PWM influence shows that the iron loss can be significantly increased due to the influence of PWM, especially when the switching frequency is low.

In the case of high precision A/D converters, carrier signal injection based position estimation methods can adopt a relatively higher injection frequency (>1/10 pulse width modulation (PWM) switching frequency), which can lead to enhanced system bandwidths. However, simultaneously, signal processing delay effects (e.g., PWM duty ratio updating delay, etc.) become prominent, resulting in large phase shifts of carrier currents. On the other hand, the high frequency (HF) resistance effects may also be enlarged. Consequently, large oscillating position estimation errors will arise for the pulsating injection method due to carrier signal distortions induced by the delay and HF resistance effects. In order to suppress the undesirable position errors, based on the theoretical analyses, a new compensation strategy of maximizing the useful inductive saliency part and minimising the resistive saliency components using a PI regulator is proposed. The proposed position error suppression method is very easy and simple to implement, and moreover can significantly improve the sensorless control performance. Furthermore, the similar compensation strategy can also be applied for the position error compensation for rotating injection method as will also be discussed in this paper. Finally, the theoretical analyses and compensation effectiveness are validated by experiments on a laboratory interior permanent magnet machine.

The sensorless control performance of pulsating carrier signal injection employing zero-sequence voltage for dual three-phase (DTP) permanent magnet synchronous machine (PMSM) drives is investigated in this paper. This method has the synergy of high bandwidth of zero-sequence voltage method and high position estimation accuracy of pulsating injection strategy. However, such a method for single three-phase PMSM exhibits large oscillation in position estimation error due to undesirable harmonic components in the zero-sequence carrier voltage, and the existing compensation methods usually require complex structures or intensive offline measurements. As for DTP-PMSM, the undesirable harmonic components for the pulsating injection can be suppressed with an optimum phase shift between the two injected high-frequency signals. Besides, a new simple measurement method of zero-sequence carrier voltage is proposed with only one voltage sensor placed between the two isolated neutral points. As a result, the rotor position estimation accuracy can be significantly improved. The experimental results verify the effectiveness of the proposed method for DTP-PMSM.

In this paper, the rotor position estimation accuracy of rotating carrier signal injection utilizing zero-sequence carrier voltage method for dual three-phase (DTP) permanent-magnet synchronous machines (PMSMs) is investigated. When utilizing the zero-sequence carrier voltage, the system bandwidth and rotor position estimation accuracy can be significantly enhanced. However, undesirable sixth harmonic estimation error occurs for single three-phase PMSM with rotating signal injection due to multiple saliency components in the zero-sequence carrier voltage. To solve such a problem, the existing methods require time-consuming offline measurements and complex structures. Fortunately, for the case of DTP-PMSM drives, thanks to the existence of additional degrees of freedom, two high-frequency signals can be injected independently for two winding sets. Therefore, by applying the optimum phase shift angle between the injected signals, a modified rotating carrier signal injection using zero-sequence voltage-based sensorless control strategy is proposed to suppress the sixth harmonic position errors. In addition, a new simple measurement method of zero-sequence carrier voltage is proposed where one voltage sensor is placed between the two isolated neutral points. The proposed method can achieve improved rotor position estimation accuracy with low computational burden. The experimental results verify the effectiveness of the proposed strategy.

The Fry gene, located on chromosome 13, is an evolutionarily conserved large protein from yeast to human. Our previous study genetically linked the Fry gene with differential susceptibility to mammary carcinogenesis, but whether Fry affects mammary gland development and function, as well as the growth of breast cancer cells, is largely unknown. To define the consequences of Fry loss in the mammary glands, we have generated mice conditionally deficient of the Fry gene in the mammary glands using the Cre-loxP recombination system. We examined multiple phenotypes with male and female homozygous Fry conditional knockout mice (Mfry) and control mice (WT), including body weight, preliminary observations (health and neurological flexes), open field locomotion, sensory abilities, auditory threshold, and glucose metabolism. The loss of Fry in the mammary glands didn't cause a significant difference in these genotypes between Mfry and WT mice. However, our data showed that Fry was required during pregnancy, while it was functionally dispensable in virgin mammary gland development. Loss of Fry led to more lateral buds, and the lobuloalveoli were smaller and showed undistended morphology in mammary glands during late pregnancy. in vitro experiment, ectopic expression of FRY could alter the morphology and significantly suppress the growth and proliferation of the breast cancer cell lines, MDA-MB-231 (ER-/PR-/HER2-, Basal-like) and BT474 (ER+/PR+/HER2+, Luminal B). The following genome-wide transcriptomic analysis of these cells suggested that FRY interacted with protein kinases relevant signaling pathways and induced massive changes in gene expression, including the activation of the Hippo/Yap pathway. Together, our data suggest that the FRY is required for mammary glands developments during pregnant periods, and affects breast cancer cell growth and proliferation.

In this paper, the non-conventional angle displacement between two sets of 3-phase windings will be analyzed and compared for the dual 3-phase fractional-slot PM machines with non-overlapping windings. Firstly, all feasible angle displacements between two sets of 3-phase windings are discussed in general for various slot/pole number combinations. Then, a dual 3-phase 24-slot/22-pole PM machine with different angle displacements (i.e. 0/15/30 deg.) is selected as an example for further in-depth investigations. It shows that under healthy condition, the electromagnetic performances of the 15 deg. angle displacement are comparable to those of the conventional counterparts. Furthermore, under 3-phase open-circuit (OC) condition, the 15 deg. angle displacement suffers less from the cross-coupling saturation effects, and thus better over-rating capability than the conventional angle displacements. Moreover, it is also found that the proposed 15 deg. angle displacement has better PM irreversible demagnetization withstand capability under 3-phase OC condition. All the theoretical analyses are verified by FE analyses on a dual 3-phase 24-slot/22-pole surface-mounted PM (SPM) machine.

A hybrid transition-metal/radical process is described that results in the addition of organozinc reagents and alkyl halides across alkenyl boron reagents in an enantioselective catalytic fashion. The reaction can be accomplished both intermolecularly and intramolecularly, providing useful product yields and high enantioselectivities in both manifolds.

Chronic arsenic exposure via drinking water has become a worldwide public health concern. In humans, inorganic arsenic (iAs) is metabolized to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) mainly mediated by arsenic (+3 oxidation state) methyltransferase (As3MT). We reported recently that N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) was involved in arsenic metabolism, and examined its interactive effect with As3MT on arsenic metabolism in vitro. To further evaluate the interactive effect of N6AMT1 and As3MT on arsenic biomethylation in humans, we conducted a human population-based study including 289 subjects living in rural villages in Inner Mongolia, China, and assessed their urinary arsenic metabolites profiles in relation to genetic polymorphisms and haplotypes of N6AMT1 and As3MT. Five N6AMT1 single nucleotide polymorphisms (SNPs; rs1003671, rs7282257, rs2065266, rs2738966, rs2248501) and the N6AMT1 haplotype 2_GGCCAT were significantly associated with the percentage of iAs (% iAs) in urine (e.g., for rs7282257, mean was 9.62% for TT, 6.73% for AA). Rs1003671 was also in a significant relationship with urinary MMA and DMA (the mean of %MMA was 24.95% for GA, 31.69% for GG; the mean of % DMA was 69.21% for GA, 59.82% for GG). The combined effect of N6AMT1 haplotype 2_GGCCAT and As3MT haplotype 2_GCAC showed consistence with the additive significance of each haplotype on % iAs: the mean was 5.47% and 9.36% for carriers with both and null haplotypes, respectively. Overall, we showed that N6AMT1 genetic polymorphisms were associated with arsenic biomethylation in the Chinese population, and its interaction with As3MT was observed in specific haplotype combinations.

Inorganic arsenic (iAs) and its high toxic metabolite, monomethylarsonous acid (MMA(III)), are able to induce malignant transformation of human cells. Chronic exposure to these chemicals is associated with an increased risk of developing multiple cancers in human. However, the mechanisms contributing to iAs/MMA(III)-induced cell malignant transformation and carcinogenesis are not fully elucidated. We recently showed that iAs/MMA(III) exposure to human cells led to a decreased level of histone acetylation globally, which was associated with an increased sensitivity to arsenic cytotoxicity. In the current study, it demonstrated that prolonged exposure to low-level MMA(III) in human urothelial cells significantly increased the expression and activity of histone deacetylases (HDACs) with an associated reduction of histone acetylation levels both globally and lysine specifically. Administration of the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), at 4 weeks after the initial MMA(III) treatment inhibited the MMA(III)-mediated up-regulation of the expression and activities of HDACs, leading to increase histone acetylation and prevention of MMA(III)-induced malignant transformation. These new findings suggest that histone acetylation dysregulation may be a key mechanism in MMA(III)-induced malignant transformation and carcinogenesis, and that HDAC inhibitors could be targeted to prevent or treat iAs-related cancers.

We have recently identified a human homolog of a fungal nuclear migration protein (hNUDC) that binds specifically with the extracellular domain of thrombopoietin receptor (Mpl). Preliminary studies with human CD34(+) cells cultured in serum-free medium and normal mice showed that hNUDC appears to act as a cytokine, triggering many of the same responses as thrombopoietin (TPO). More intriguingly, recent data gained using a NIH 3T3 system have demonstrated that hNUDC exerts its biological activities through activation of Mpl. In this study, we further compared the biological functions of hNUDC with TPO in an EPO-dependent UT-7 cell line that was engineered to express the thrombopoietin receptor (Mpl). These Mpl-expressing cells following stimulation by either hNUDC or TPO exhibited overlapping patterns of megakaryocytic proliferation and differentiation, manifested by cell morphological change, polyploidy and expression of CD41(+). Similar with TPO, hNUDC induced a sustained activation of the extracellular signal-regulated protein kinases-1 and -2 (ERK1/2) as well as p38 mitogen-activated kinase (p38 MAPK) pathways and these activations were inhibited in the presence of PD98059 or SB203580. Further evidence is provided that PD98059 or SB203580 inhibited hNUDC- or TPO-induced cell proliferation and differentiation, suggesting that ERK1/2 and p38 MAPK pathways are necessary in megakaryocyte development.

Human NUDC (hNUDC) has been previously described as a human homolog of a fungal nuclear migration protein. It is a multifunctional interactive protein that forms an association with the microtubule motor complex in a variety of cells. Our recent studies demonstrated that hNUDC could bind specifically to the thrombopoietin receptor (Mpl) and suggest a potential role for hNUDC in megakaryocytopoiesis and thrombopoiesis. The present study is designed to define its biological activity. We demonstrate that the recombinant hNUDC significantly increases megakaryocyte maturation in serum-free liquid-cultured human CD34+ cells and stimulates colony formation in serum-free semi-solid cultures. Flow cytometry analyses also confirm the stimulatory effect of hNUDC on megakaryocyte polyploidization and in vitro platelet production. In vivo experiments further demonstrate that the administration of hNUDC substantially enhance the number of circulating platelets in normal mice. J. Cell. Biochem. 98: 429–439, 2006. © 2006 Wiley-Liss, Inc.

Mutants of class I enolpyruvylshikimate 3-phosphate synthase (EPSPS) with resistance to glyphosate were produced in a previous study using the staggered extension process with aroA genes from S. typhimurium and E. coli. Two of these mutants shared a common amino acid substitution, T42M, near the hinge region between the large globular domains of EPSPS. Using site-directed mutagenisis, we produced the T42M mutants without the other amino acid changes of the original mutants. The T42M substitution alone produced enzymes with a 9- to 25-fold decreased K(m)[PEP] and a 21- to 26-fold increased K(i)[glyphosate] compared to the wild-type enzymes. These results provide more testimony for the powerful approach for protein engineering by the combination of directed evolution and rational design.

This study intends to make a comparative analysis of the accounting standards for consolidation and investment in the three Chinese regions, i.e. the Mainland, Taiwan and Hong Kong, against the international accounting standards and the U.S. GAAP in order to facilitate more informative financial reporting. We also discuss the disclosure and supervisory requirements for equity investments. Due to the differences in the historical backgrounds and stages of economic development among the three regions, it is inevitable and necessary for the regions to maintain their respective characteristics in the accounting standards. Our analysis would generate some insight into how the three regions could reduce unnecessary differences between accounting standards in order to provide international investors with better and more reliable business information for decision making.

The binding of human thrombopoietin to the extracellular domain of its receptor MPL prompts a cascade transduction of intracellular signals, leading to the development of megakaryocyte precursors and the production of circulating platelets. We have used a yeast two-hybrid system to reveal, via in vivo interactions between different deletion constructs of MPL and thrombopoietin, that the extracellular subunit 1 of MPL is the ligand binding site and the N-terminal domain of thrombopoietin alone is sufficient for the binding. The extracellular portion of MPL was heterologously expressed in E. coli and its specific affinity with thrombopoietin was visualized in vitro by resonance mirror biosensor technique.

N-acetylchitooligosaccharides of degree of polymerization 6 to 8 elicit the synthesis of (1→3)-β-glucan endohydrolase (EC 3.2.1.39) activity in suspension-cultured cells derived from immature barley (Hordeum vulgare) embryos. Corresponding de-acetylated chitooligosaccharides have no effect. Concentrations of N-acetylchitoheptaose in the 200 nM range are sufficient to elicit the response. A 2- to 3-fold increase in (1→3)-β-glucanase activity is detected 24 h after the addition of the oligosaccharide. Non-denaturing gel electrophoresis, coupled with the in situ detection of (1→3)-β-glucanase activity in the gels, has enabled the separation of specific isoforms of the enzyme. The increase in (1→3)-β-glucanase activity following N-acetylchitoheptaose induction is attributable predominantly to enhanced levels of isoenzyme GII, although the barley (1→3)-β-glucanase gene family encodes at least seven different isoforms of the enzyme. Northern hybridization analyses with gene-specific probes confirmed the presence of mRNA encoding isoenzyme GII as the major mRNA in cells treated with the oligosaccharide elicitor. The results therefore demonstrate a specific induction of the (1→3)-β-glucanase isoenzyme GII gene following stimulation of barley cells with oligosaccharides of fungal cell wall origin, and further suggest that a plant's response to microbial attack involves transcription of quite specific members of the gene families that encode pathogenesis-related proteins.

Objectives Overexpression of hNUDC, a member of the nuclear distribution protein family, reduces cell population growth in prostate cancer cell lines, concurrent with induced morphological change and enhanced polyploidization. These phenomena are also closely associated with terminal phases of megakaryocyte maturation. Materials and methods In Dami cells, MTT and trypan blue assays were used to investigate cell viability and proliferation effects of hNUDC, and flow cytometry was used to analyse cell cycle and DNA content. Real-time RT-PCR was employed to detect mRNA expression. Activations of caspase-3, ERK, Akt and Stat-5 were determined by immunoblotting. May-Grunwald–Giemsa staining was performed to reveal cell morphology. Results and conclusion Functional studies using adenovirus-mediated hNUDC overexpression led to inhibition of megakaryocyte proliferation via cell cycle arrest in G2/M transition phase. This process could have been be mediated by upregulation of p21 and downregulation of its downstream targets, including cyclin B1, cyclin B2 and c-myc. Enhanced apoptosis in turn ensued, characterized by increased caspase-3 activation, upregulation of pro-apoptotic Bax and downregulation of anti-apoptotic Bcl-2. Furthermore, hNUDC overexpression elevated the level of megakaryocyte maturation, associated with increased polyploidy, cell morphological changes and increased expression of cell surface differentiation markers, including CD10, CD44, CD41 and CD61. Our results further suggest that the ERK signalling pathway was involved in hNUDC overexpression-induced apoptosis. Taken together, this study provides experimental evidence for overexpression of hNUDC in Dami cells and suggests that activation of apoptotic machinery may be involved in megakaryocytic differentiation.

The thrombopoietin (TPO) receptor (Mpl) belongs to the family of ligand-dependent cytokine receptors and plays a functional role in regulating platelet production. The signaling capacity largely depends on the binding of TPO to the extracellular domains of the TPO receptor (Mpl-EC). Because the expression level of Mpl in human tissue is very low, studies on the functional and spatial characteristics of its ligand-binding sites have been limited. In the present study, we report the expression and purification of Mpl-EC as a fusion with the maltose-binding protein (MBP), designated MBP-Mpl-EC. MBP-Mpl-EC was expressed in the cytoplasm of Escherichia coli as a soluble fusion protein. Specific binding of TPO to purified MBP-Mpl-EC was demonstrated by a dot-blot assay and surface plasmon resonance. We conclude that bacterial expression of MBP-Mpl-EC yields large amounts of protein with correct folding and that it can be used for further structure and function analyses.

We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.

Ustilago maydis secretes ferrichrome-type siderophores, ferric-ion-binding compounds, in response to iron starvation. TA2701, a non-enterobactin-producing, non-ferrichrome-utilizing mutant of Salmonella typhimurium LT-2, was employed as a biological indicator in a novel screening method to isolate three N-methyl-N'-nitro-N-nitrosoguanidine-induced U. maydis mutants defective in the regulation of ferrichrome-type siderophore biosynthesis. These mutants displayed a constitutive phenotype; they produced siderophores in the presence of iron concentrations that would typically repress siderophore synthesis in wild-type strains. A 4.8-kb fragment of U. maydis genomic DNA capable of restoring normal regulation of siderophore biosynthesis in the constitutive mutants was identified. This segment of DNA contains an intronless open reading frame that specifies a protein of 950 amino acids containing two finger motifs similar to those found in the erythroid transcription factor GATA-1. Disruption of this open reading frame in a wild-type strain gave rise to cells that produced siderophores constitutively. Genetic studies indicated that the disruption mutation was allelic to the chemically induced mutations, confirming that the structural gene for a regulator rather than a suppressor gene had been cloned. Northern (RNA) analysis of the gene revealed a 4.2-kb transcript that is expressed constitutively at low levels in wild-type cells. The data support the hypothesis that this gene, which we designate urbs1 (Ustilago regulator of biosynthesis of siderophores), acts directly or indirectly to repress biosynthesis of siderophores in U. maydis.

The human homolog of a fungal nuclear migration protein (hNUDC) has recently been shown in in vitro studies to overlap in function with the cytokine factor thrombopoietin (TPO) in its ability to induce human megakaryocyte development. In the present study, we sought to confirm the hypothesis that combination of TPO and NUDC may result in additive or synergistic effects on megakaryocyte maturation in bone marrow. For this purpose, murine bone marrow cells were stimulated with murine NUDC (mNUDC), either alone or in conjunction with murine thrombopoietin (mTPO), in serum-free medium. Studies showed that mNUDC + mTPO treatments resulted in the greatest number of colony-forming unit of megakaryocytes. Concomitantly, mNUDC + mTPO enhanced expression of CD61 and elicited the largest number of megakaryocytes, with higher ploidy and larger size. In addition, in vivo experiments revealed an elevation of platelet levels in normal and thrombocytopenic mice that had been administered mTPO + mNUDC. Moreover, mRNA levels of mNUDC and murine thrombopoietin receptor were much higher than the levels of mTPO mRNA at two different phases of normal murine megakaryocyte maturation. Furthermore, levels of ERK1/2 and Akt phosphorylation are higher with mNUDC in combination with mTPO. This study demonstrates that the combination of mTPO and mNUDC provides additive induction of megakaryocyte maturation in vitro and platelet production in vivo.

Thrombopoietin (TPO) and its receptor (Mpl) have long been associated with megakaryocyte proliferation, differentiation, and platelet formation. However, studies have also shown that the extracellular domain of Mpl (Mpl-EC) interacts with human (h) NUDC, a protein previously characterized as a human homolog of a fungal nuclear migration protein. This study was undertaken to further delineate the putative binding domain on the Mpl receptor. Using the yeast two-hybrid system assay and co-immunoprecipitation, we identified that within the Mpl-EC domain 1 (Mpl-EC-D1), amino acids 102-251 were strongly involved in ligand binding. We subsequently expressed five subdomains within this region with T7 phage display. Enzyme-linked immunosorbent binding assays identified a short stretch of peptide located between residues 206 and 251 as the minimum binding domain for both TPO and hNUDC. A series of sequential Ala replacement mutations in the region were subsequently used to identify the specific residues most involved in ligand binding. Our results point to two hydrophobic residues, Leu(228) and Leu(230), as having substantial effects on hNUDC binding. For TPO binding, mutations in residues Asp(235) and Leu(239) had the largest effect on binding efficacy. In addition, deletion of the conservative motif WGSWS reduced binding capacity for hNUDC but not for TPO. These separate binding sites on the Mpl receptor for TPO and hNUDC raise interesting implications for the cytokine-receptor interactions.

A new member of the barley (1--3)-beta-glucan glucanohydrolase family of enzymes has been purified from extracts of germinated grain and young seedlings by fractional precipitation with ammonium sulphate, ion-exchange chromatography, chromatofocussing and gel-filtration chromatography. The enzyme, which has been designated (1--3)-beta-glucanase isoenzyme GIII, is a basic protein with an apparent molecular mass of 32 000 Da. Oligosaccharide products released by the enzyme during hydrolysis of the (1--3)-beta-glucan, laminarin, indicate that the enzyme is an endohydrolase. A 2349-bp fragment of barley genomic DNA has been isolated and identified as the gene encoding the (1--3)-beta-glucanase isoenzyme GIII. The open reading frame encoding the isoenzyme is interrupted by a single intron of 180 bp that splits a codon in the putative signal-peptide region. Northern-blot analyses with gene-specific probes indicate that the (1--3)-beta-glucanase isoenzyme GIII mRNA accumulates in developing leaves; no mRNA transcripts were detected in the aleurone or scutellum of germinated grain, or in mature vegetative tissues. Although plant (1--3)-beta-glucanases are generally classified as 'pathogenesis-related' proteins, the physiological function of the barley (1--3)-beta-glucanase isoenzyme GIII is unclear.

Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.

Drosomycin(Drs) encoding an inducible 44-residue antifungal peptide is clustered with six additional genes,Dro1, Dro2, Dro3, Dro4, Dro5,andDro6, forming a multigene family on the 3L chromosome arm inDrosophila melanogaster. To get further insight into the regulation of each member of the drosomycin gene family, here we investigated gene expression patterns of this family by either microbe-free injury or microbial challenges using real time RT-PCR. The results indicated that among the sevendrosomycingenes,Drs, Dro2, Dro3, Dro4,andDro5showed constitutive expressions. Three out of five,Dro2, Dro3,andDro5, were able to be upregulated by simple injury. Interestingly,Drsis an only gene strongly upregulated whenDrosophilawas infected with microbes. In contrast to these five genes,Dro1andDro6were not transcribed at all in either noninfected or infected flies. Furthermore, by5′rapid amplification of cDNA ends, two transcription start sites were identified inDrsandDro2, and one inDro3, Dro4,andDro5. In addition, NF-κB binding sites were found in promoter regions ofDrs, Dro2, Dro3,andDro5, indicating the importance of NF-κB binding sites for the inducibility ofdrosomycingenes. Based on the analyses of flanking sequences of each gene inD. melanogasterand phylogenetic relationship ofdrosomycinsinD. melanogasterspecies-group, we concluded that gene duplications were involved in the formation of the drosomycin gene family. The possible evolutionary fates ofdrosomycingenes were discussed according to the combining analysis of gene expression pattern, gene structure, and functional divergence of these genes.