A cell penetrating heme oxygenase protein protects heart graft against ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury is an unavoidable barrier that significantly affects outcome of solid organ transplantation. Here, we establish a protein transduction system to extend graft preservation time and to prevent I/R injury in heart transplantation. We generated a recombinant heme oxygenase-1 (HO-1) protein containing a modified protein transduction domain (PTD). PTD could cross cover cell membrane and carry target molecule to parenchymal cells of cold-preserved heart grafts. The newly generated PTD-HO-1 protein localized mainly in subcellular membrane organelle and nucleus after delivery that significantly prolonged cold preservation of heart grafts. This effect was associated with significantly less endothelial cell activation, less neutrophil and macrophage infiltration in PTD-HO-1-transduced heart grafts after reperfusion as compared with controls. In addition, transduction of PTD-HO-1 protein to heart graft significantly suppressed the I/R injury-associated myocardiocyte apoptosis. The infarct areas of heart graft after I/R injury were significantly reduced after PTD-HO-1 protein treatment. We show here for the first time that PTD can maintain its biological activities during cold preservation. Transduction of cell penetrating HO-1 protein significantly prolongs the cold preservation time and protects the graft from the I/R injury. This approach represents a novel method for the improvement of the overall outcome of organ transplantation.

Two-dimensional model of low Mach number vortex sound generation in a lined duct.

The sound generated by a vortex moving across a duct section lined with porous materials and the corresponding vortex dynamics are studied numerically in the present investigation. The combined effects of the effective fluid density, the flow resistance, the length, and the thickness of the porous linings on the vortex dynamics and sound generation are examined in detail. Results show that stronger sound radiation will take place when the length and the thickness of the porous linings are increased or when the effective fluid density is reduced. The flow resistance can only result in stronger sound radiation within a range whose width depends on the above-mentioned system parameters. Such sound amplification cannot be achieved when the initial vortex height gets closer and closer to the duct centerline. The present results also indicate the strong correlation between vortex acceleration and the sound radiation under the actions of the porous linings.

Ribosomal DNA replication fork barrier and HOT1 recombination hot spot: shared sequences but independent activities.

In the ribosomal DNA of Saccharomyces cerevisiae, sequences in the nontranscribed spacer 3' of the 35S ribosomal RNA gene are important to the polar arrest of replication forks at a site called the replication fork barrier (RFB) and also to the cis-acting, mitotic hyperrecombination site called HOT1. We have found that the RFB and HOT1 activity share some but not all of their essential sequences. Many of the mutations that reduce HOT1 recombination also decrease or eliminate fork arrest at one of two closely spaced RFB sites, RFB1 and RFB2. A simple model for the juxtaposition of RFB and HOT1 sequences is that the breakage of strands in replication forks arrested at RFB stimulates recombination. Contrary to this model, we show here that HOT1-stimulated recombination does not require the arrest of forks at the RFB. Therefore, while HOT1 activity is independent of replication fork arrest, HOT1 and RFB require some common sequences, suggesting the existence of a common trans-acting factor(s).

Vortex sound under the influence of a piecewise porous material on an infinite rigid plane.

The vortex dynamics and the sound generation by an inviscid vortex in the presence of a finite length porous material on an otherwise rigid plane are studied numerically in the present study in an attempt to understand the sound generation near the surface of a wall lining in a lined duct. The combined effects of the effective fluid density and flow resistance inside the porous material, and the length and thickness of the porous material on the sound generation process are examined in detail. Results obtained demonstrate the sound pressure is longitudinal dipole and show how seriously the above-mentioned parameters are affecting the vortex sound pressure under the influence of the porous material.

Next-generation sequencing with a myeloid gene panel in core-binding factor AML showed KIT activation loop and TET2 mutations predictive of outcome.

Clinical outcome and mutations of 96 core-binding factor acute myeloid leukemia (AML) patients 18-60 years old were examined. Complete remission (CR) after induction was 94.6%. There was no significant difference in CR, leukemia-free-survival (LFS) and overall survival (OS) between t(8;21) (N=67) and inv(16) patients (N=29). Univariate analysis showed hematopoietic stem cell transplantation at CR1 as the only clinical parameter associated with superior LFS. Next-generation sequencing based on a myeloid gene panel was performed in 72 patients. Mutations in genes involved in cell signaling were associated with inferior LFS and OS, whereas those in genes involved in DNA methylation were associated with inferior LFS. KIT activation loop (AL) mutations occurred in 25 patients, and were associated with inferior LFS (P=0.003) and OS (P=0.001). TET2 mutations occurred in 8 patients, and were associated with significantly shorter LFS (P=0.015) but not OS. Patients negative for KIT-AL and TET2 mutations (N=41) had significantly better LFS (P<0.001) and OS (P=0.012) than those positive for both or either mutation. Multivariate analysis showed that KIT-AL and TET2 mutations were associated with inferior LFS, whereas age ⩾40 years and marrow blast ⩾70% were associated with inferior OS. These observations provide new insights that may guide better treatment for this AML subtype.

Binding of polylysine to GroEL. Inhibition of the refolding of mMDH.

Luminescence techniques have been used to investigate the interaction of GroEL with polylysine tagged with a fluorescent probe. The fluorescence emitted by anthraniloyl-polylysine, upon excitation at 320 nm, is enhanced by the addition of stoichiometric amounts of GroEL. The equilibrium dissociation constant of the complex (Kd=50 nM) was determined by fluorometric titrations. The rate and extent of recovery of the catalytic activity of denatured mitochondrial malate dehydrogenase, assisted by GroEL, is influenced by either polylysine or anthraniloyl-polylysine. It is suggested that interaction of the positively charged poly-amino acid with the apical domain of GroEL prevents binding of the unfolded protein substrate.

Rapid inhibition of c-myc gene expression by a glucocorticoid in the avian oviduct.

The glucocorticoid dexamethasone (DEX) causes a rapid, reversible reduction in c-myc mRNA level in the oviducts of estrogen-treated, immature chickens. The c-myc mRNA level begins to decrease by 5 min after injection of 0.5 mg DEX, reaches a minimum of 10% of the control value by 30 min, and returns to 30-40% of the control value by 4 h post injection. This rapid effect of DEX on the c-myc mRNA level occurs in both diethylstilbestrol-stimulated and diethylstilbestrol-withdrawn oviducts. The effect is dose dependent, with reduction of the c-myc mRNA measured with as little as 10 micrograms DEX injection (0.03 micrograms/g BW). The effect of the steroid is gene specific with H2B histone mRNA displaying a significantly reduced response. The effect is also tissue specific with liver displaying an increase of 170% of control values in c-myc mRNA level by 30 min after injection of 0.5 mg DEX. The reduction of avian oviduct c-myc mRNA levels by DEX may play a role in glucocorticoid inhibition of cell proliferation in this tissue. The rapidity of the steroid effects on c-myc expression makes it likely that the steroid-induced reduction of c-myc mRNA levels represents a direct primary action of the steroid-receptor complex on the c-myc gene expression.

Porcine pyridoxal kinase c-DNA cloning, expression and confirmation of its primary sequence.

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.

Rapid inhibition of the c-jun proto-oncogene expression in avian oviduct by estrogen.

The c-jun proto-oncogene codes for an important component of the AP-1 transcription factor complex which regulates the expression of many genes. This paper demonstrates that the steady state c-jun mRNA level in the avian oviduct is markedly decreased to 50% of control values within 30 minutes after estrogen injection into the animals. In the avian liver, the level is rapidly increased over 10 fold. Nuclear run-off transcription analysis demonstrates that the decrease in the c-jun mRNA levels in the avian oviduct occurs at least in part at the level of transcription. These changes are reproducible in repeat experiments. This response in the avian oviduct is unique since 1) it is the first demonstration of a steroid effect on c-jun expression in any animal system, 2) the changes in c-jun mRNA occur much more rapidly than most steroid responsive genes, and 3) it is the rare demonstration of an estrogen inhibition of the expression of a gene. A role for the c-jun proto-oncogene as an early regulatory gene in the cascade model for steroid action is proposed.

Development of 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-phenylethyl)-5-benzofuranol (L-670,630) as a potent and orally active inhibitor of 5-lipoxygenase.

Leukotrienes are potent biological mediators of allergic and inflammatory diseases and are derived from arachidonic acid through the action of the 5-lipoxygenase. In this study, the syntheses and comparative biological activities of three series of 2,3-dihydro-2,6-disubstituted-5-benzofuranols with various substituents on position 3 are described. Compounds from each series were evaluated for their ability to inhibit the production of leukotriene B4 (LTB4) in human peripheral blood polymorphonuclear (PMN) leukocytes and the 5-lipoxygenase reaction in cell-free preparations from rat PMN leukocytes. The structure-activity relationships of each series in vitro and in vivo are presented. The bioavailability, metabolism, and toxicity profile of each series are discussed. The series with no substituent at position 3 was the most potent and among the compounds in that series 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-phenylethyl)-5-benzofuranol (46, L-670,630) was chosen for further development.

Synthesis and structure-activity relationships of a novel class of 5-lipoxygenase inhibitors. 2-(Phenylmethyl)-4-hydroxy-3,5-dialkylbenzofurans: the development of L-656,224.

The synthesis of a series of 2-(phenylmethyl)-4-hydroxy-3,5-dialkylbenzofurans and their inhibitory effects against leukotriene biosynthesis and 5-lipoxygenase activity in vitro are described. Many compounds in this series were found to be potent inhibitors of LTB4 production by human polymorphonuclear leukocytes with IC50 values ranging from 7 to 100 nM. Structure-activity relationships of the series are presented. Within this series, 2-[(4'-methoxyphenyl)methyl]-4-hydroxy-3-methyl-5-propyl-7-chlorobenz ofuran (L-656,224) showed extremely potent activity, inhibiting leukotriene biosynthesis in intact human leukocytes (IC50 = 11 nM), as well as the 5-lipoxygenase reaction catalyzed by cell-free preparations from rat leukocytes (IC50 = 36 nM), human leukocytes (IC50 = 0.4 microM), and the purified enzyme from porcine leukocytes (IC50 = 0.4 microM). The compound also shows oral activity in a number of animal models in vivo.

Cyclooxygenase-2 inhibitors. Synthesis and pharmacological activities of 5-methanesulfonamido-1-indanone derivatives.

The recent discovery of an alternative form cyclooxygenase (cyclooxygenase-2, COX-2), which has been proposed to play a significant role in inflammatory conditions, may provide an opportunity to develop anti-inflammatory drugs with fewer side effects than existing non-steroidal anti-inflammatory drugs (NSAIDs). We have now identified 6-[(2,4-difluorophenyl)-thio]-5-methanesulfonamido-1-indanone++ + (20) (L-745,337) as a potent, selective, and orally active COX-2 inhibitor. The structure-activity relationships in this series have been extensively studied. Ortho- and para-substituted 6-phenyl substitutents are optimal for in vitro potency. Replacement of this phenyl ring by a variety of heterocycles gave compounds that were less active. The methanesulfonamido group seems to be the optimal group at the 5-position of the indanone system. Compound 20 has an efficacy profile that is superior or comparable to that of the nonselective COX inhibitor indomethacin in animal models of inflammation, pain, and fever and appears to be nonulcerogenic within the dosage ranges required for functional efficacy. Although 20 and its oxygen linkage analog 2 (flosulide) are equipotent in the in vitro assays, compound 20 is more potent in the rat paw edema assay, has a longer t1/2 in squirrel monkeys, and seems less ulcergenic than 2 in rats.

Inhibition of human leukocyte 5-lipoxygenase by a 4-hydroxybenzofuran, L-656,224. Evidence for enzyme reduction and inhibitor degradation.

Detailed studies of the interaction of L-656,224 (2-[(4'-methoxyphenyl)methyl]-3-methyl-4-hydroxy-5-propyl-7- chlorobenzofuran) with 5-lipoxygenase were conducted using the enzymes from human and pig leukocytes. L-656,224 was a potent inhibitor of these 5-lipoxygenases although its efficiency varied with enzyme concentration. L-656,224 also stimulated the pseudoperoxidase activity of 5-lipoxygenase as measured by the consumption of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD), indicating that this compound can reduce the enzyme. Furthermore the inhibitor was degraded rapidly by both cell-free leukocyte extracts and purified 5-lipoxygenase after incubation with 13-HPOD, ATP and calcium ions. The degradation of L-656,224 was also observed during inhibition of the lipoxygenase reaction and occurred mainly after the initial lag phase of the reaction when hydroperoxides begin to accumulate. A single major radioactive product was formed after incubation of [3H]L-656,224 with purified 5-lipoxygenase in the presence of 13-HPOD. This product was unstable and could not be isolated. During the course of the pseudoperoxidase reaction, [3H]L-656,224 covalently labelled the enzyme, suggesting that a chemically reactive species had been formed. These data are consistent with the hypothesis that L-656,224 reduces the oxidized form of the 5-lipoxygenase to an inactive form, with degradation of the inhibitor and regeneration of the active enzyme with hydroperoxides.

Antimicrobial action of compound 48/80--II mechanism of action.

The mixture of compounds called compound 48/80 had been shown to have antimicrobial activity against a wide variety of microorganisms. In this paper it is shown that its primary site of attack appears to be on the membrane of the cell. In its presence, Tetrahymena became much more sensitive to osmotic stress, and alpha-methylglucose was rapidly released from preloaded Escherichia coli cells. The drug also had effects on cell viability, respiration, cell division, and the release of material absorbing at 260 nm. In general, its effects paralleled those of polymyxin B, although its structure is quite different except for the presence of amino groups and hydrophobic regions in both molecules. The activity of 48/80 was not due to detergent-like, surface-active properties and was antagonized by magnesium and other cations and by phosphatidylserine. Purification of the active principle might provide a relatively simple and readily modifiable probe of membrane function and possibly a new family of useful antimicrobial compounds.

Autologous bone marrow transplantation for patients with acute myeloid leukaemia: prospective follow-up study.

OBJECTIVE: To study the use of autologous bone marrow transplantation to treat acute myeloid leukaemia when complete remission had been achieved and when no human leukocyte antigen matched related donor was available. DESIGN: Prospective follow-up study. SETTING: Government hospital, Hong Kong. PATIENTS: Eight patients (median age, 34 years [range, 16-45 years]) with acute myeloid leukaemia in whom complete remission had been achieved. INTERVENTION: Conditioning regimen of carmustine, amsacrine, etoposide VP-16, cytarabine, and infusion of unpurged marrow. MAIN OUTCOME MEASURES: Median time taken to reach neutrophil and platelet counts of > or =0.5 x 10(9) /L and > or = x 10(9) /L, respectively; mortality and relapse rates; and follow-up regimens used. RESULTS: Engraftment was successfully achieved in all patients and there were no early procedure-related mortalities. The median times required to reach a neutrophil count of > or =0.5 x 10(9) /L and a platelet count of > or =20 x 10(9) /L were 30 days (range, 18-36 days) and 38 days (range, 15-53 days), respectively. The median duration of hospital stay was 37 days (range, 25-43 days). Two patients died of a relapse of leukaemia at 6 and 9 months post-transplantation. Two patients experienced relapses: one at 8 months post-transplantation, for which conventional chemotherapy was restarted, and one at 18 months; treatment with all-trans-retinoic acid and conventional chemotherapy achieved a third complete remission in the latter patient, who had acute promyelocytic leukaemia. Continuous remission has been achieved in four of the eight patients after a median follow-up duration of 26 months (range, 6-43 months). CONCLUSION: Autologous bone marrow transplantation is an acceptable treatment for patients with acute myeloid leukaemia who lack a human leukocyte antigen-matched related donor.

Academic background of oral and maxillofacial specialty trainees in the United Kingdom.

INTRODUCTION: The academic profile of a specialty can be measured in a number of ways. In the selection process for entry into higher training in oral & maxillofacial surgery (OMFS) and for interface fellowships in surgery trainees are awarded points for papers published, presentations, teaching and learning, grants and higher degrees. General information about career development can provide trainers, and trainees, with information and guidance. METHODS: A web-based questionnaire was developed and distributed via electronic mailing lists to all OMFS specialist trainees. Basic demographic information was collected. Previous, current and future career plans were questioned, as was academic background in terms of publications, presentations, prizes and research grants as well as further degrees and examinations. RESULTS: One hundred and five OMFS specialty registrar trainees (StR) replied (76.6% response rate). 83.3% were male and the average age of all trainees was 37 years old. 74.7% obtained a training post on the first application. 62.6% of trainees were keen to practice in trauma surgery. 76.6% were keen to undertake a fellowship. 20.9% were keen to be involved in academia (teaching) and 9.9% in academia (research). 22.1% of trainees had obtained grants. CONCLUSION: Those involved in appointing to training programmes will now be able to see the level of competition. Future applicants to training programmes in oral and maxillofacial surgery in the United Kingdom are now aware of the level of competition. OMFS is not immune to the 'academic crisis' that exists in other surgical specialties, and the completion of higher degrees and entry in to academic careers should be encouraged and supported among trainees with an interest.

Localization of burn mark under an abnormal topography on MOSFET chip surface using liquid crystal and emission microscopy tools.

This article focuses on the localization of burn mark in MOSFET and the scanning electron microscope (SEM) inspection on the defect location. When a suspect abnormal topography is shown on the die surface, further methods to pin-point the defect location is necessary. Fault localization analysis becomes important because an abnormal spot on the chip surface may and may not have a defect underneath it. The chip surface topography can change due to the catastrophic damage occurred at layers under the chip surface, but it could also be due to inconsistency during metal deposition in the wafer fabrication process. Two localization techniques, liquid crystal thermography and emission microscopy, were performed to confirm that the abnormal topography spot is the actual defect location. The tiny burn mark was surfaced by performing a surface decoration at the defect location using hot hydrochloric acid. SEM imaging, which has the high magnification and three-dimensional capabilities, was used to capture the images of the burn mark.

Fault diagnosis of the polypropylene production process (UNIPOL PP) using ANFIS.

The performance of a chemical process plant can gradually degrade due to deterioration of the process equipment and unpermitted deviation of the characteristic variables of the system. Hence, advanced supervision is required for early detection, isolation and correction of abnormal conditions. This work presents the use of an adaptive neuro-fuzzy inference system (ANFIS) for online fault diagnosis of a gas-phase polypropylene production process with emphasis on fast and accurate diagnosis, multiple fault identification and adaptability. The most influential inputs are selected from the raw measured data sets and fed to multiple ANFIS classifiers to identify faults occurring in the process, eliminating the requirement of a detailed process model. Simulation results illustrated that the proposed method effectively diagnosed different fault types and severities, and that it has a better performance compared to a conventional multivariate statistical approach based on principal component analysis (PCA). The proposed method is shown to be simple to apply, robust to measurement noise and able to rapidly discriminate between multiple faults occurring simultaneously. This method is applicable for plant-wide monitoring and can serve as an early warning system to identify process upsets that could threaten the process operation ahead of time.