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Hairy root cultures of Gynostemma pentaphyllum were established by infecting leaf discs with Agrobacterium rhizogenes. The dry biomass of hairy roots grown in MS medium for 49 days was 7.3 g l(-1) with a gypenoside content of 38 mg g(-1) dry wt.

Bovine ephemeral fever (BEF) is a viral disease of cattle. A flow type quartz crystal microbalance (QCM) immunosensor was developed for the real time determination BEF virus (BEFV) that is suitable for clinical point-case diagnosis. Self-assembled monolayer (SAM) of thiols and sulphides by the cystamine-glutaraldehyde method was used for the immobilization of BEFV monoclonal antibody on the gold surface of a quartz crystal microbalance (QCM). A positive correlation was found between the virus concentration and frequency changes (R(2)=0.9962) on this QCM system. The reproducible rates for the 50 and 10mug/mL samples were 4 and 13.9%, respectively. There was no interference from non-specifically adsorbed phage. Using this flow type QCM immunosensor, BEFV could specifically be detected with sensitivity comparable to a conventional enzyme-linked immunosorbent assay (ELISA). The measurement could be obtained directly, within several minutes, rather than hours as required visualizing the results of ELISA. In addition, the observation of reproducible and constant changes after successive additions of BEFV suggests that a QCM immunosensor in a flow cell could be developed for automated or continuous real time operation.

The promyelocytic leukemia gene (PML), which is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL), encodes a multifunctional protein involved in several important cellular functions. Herein, we demonstrate that PML is localized to centrosomes and that PML deficiency leads to centrosome amplification. By using PML isoform-specific antibodies, we found PML3-specific association with the centrosome and the pole of the mitotic spindle. PML3 deficiency leads to dysregulation of the centrosome duplication checkpoint. Furthermore, PML3 physically interacts with Aurora A and regulates its kinase activity. Specific knockdown of PML3 activates Cdk2/cyclin kinase activity. The results of this study implicate a direct role for PML3 in the control of centrosome duplication through suppression of Aurora A activation to prevent centrosome reduplication.

The translocation t(8;21)(q22;q22) in acute myeloid leukemia (AML) results in the expression of the fusion protein RUNX1/MTG8, which in turn recruits histone deacetylases (HDAC) to silence RUNX1 target genes [e.g., interleukin-3 (IL-3)]. We previously reported that expression of the RUNX1/MTG8 target gene IL-3 is synergistically restored by the combination of inhibitors of HDACs (i.e., depsipeptide) and DNA methyltransferases (DNMT; i.e., decitabine) in RUNX1/MTG8-positive Kasumi-1 cells. Thus, we hypothesized that DNMT1 is also part of the transcriptional repressor complex recruited by RUNX1/MTG8. By a chromatin immunoprecipitation assay, we identified a RUNX1/MTG8-DNMT1 complex on the IL-3 promoter in Kasumi-1 cells and in primary RUNX1/MTG8-positive AML blasts. The physical association of RUNX1/MTG8 with DNMT1 was shown by coimmunoprecipitation experiments. Furthermore, RUNX1/MTG8 and DNMT1 were concurrently released from the IL-3 promoter by exposure to depsipeptide or stabilized on the promoter by decitabine treatment. Finally, we proved that RUNX1/MTG8 and DNMT1 were functionally interrelated by showing an enhanced repression of IL-3 after coexpression in 293T cells. These results suggest a novel mechanism for gene silencing mediated by RUNX1/MTG8 and support the combination of HDAC and DNMT inhibitors as a novel therapeutic approach for t(8;21) AML.

Thermostable cellulase was produced by Streptomyces sp. T3-1 grown in a 50-l fermenter. Maximum cellulase activity was attained on the fourth day when agitation speeds and aeration rates were controlled at 300 rpm and 0.75 vvm, respectively. Maximum enzyme activities were: 148 IU CMCase ml(-1), 45 IU Avicelase ml(-1), and 137 IU beta-glucosidase ml(-1) with productivity of 326 IU l(-1) h(-1), which were 10-32% higher than the values obtained in shake-flask cultures.

A moderately thermophilic aerobic bacterium, strain 25-4T, was isolated from a hot spring at Baekdoo Mountain in Korea. The cells were Gram-negative, motile rods each having a polar flagellum. Analysis of the 16S rRNA gene sequence indicated that the strain represented a new lineage within the family 'Xanthomonadaceae' of the 'Gammaproteobacteria', being most closely related to the genera Thermomonas, Xanthomonas, Luteimonas, Pseudoxanthomonas, Stenotrophomonas and Xylella and having 16S rRNA gene sequence similarities to the most related species of the genera of between 92.9 and 94.4 %. The strain contained Q-8 as the major isoprenoid quinone and had a fatty acid profile with predominant iso-branched fatty acids. Growth occurred at pH 6.0-10, with an optimum at pH 9.0, and at 25-53 degrees C, with an optimum at 47 degrees C. The G+C content of the genomic DNA was 50.7 mol%. On the basis of phylogenetic analyses and its phenotypic characteristics, strain 25-4T belongs to a new genus, Silanimonas gen. nov., within the 'Gammaproteobacteria'. The sole species of this genus is Silanimonas lenta sp. nov. (type strain, 25-4T=DSM 16282T=KCTC 12236T).

The repellency of fennel (Foeniculum vulgare Miller)-containing products (5% aerosol and 8% cream) against mosquitoes was compared with those of citronella oil, geranium oil and deet, as well as three commercial repellents, Baby Keeper cream containing IR3535, MeiMei cream containing citronella and geranium oils, and Repellan S aerosol containing 19% N,N-diethyl-m-toluamide (deet) under laboratory and field conditions. In a laboratory study with female Aedes aegypti (L), fennel oil exhibited good repellency in a release-in-cage test and repellency in skin and patch tests of the oil was comparable with those of citronella and geranium oils. In paddy field tests with five human volunteers, 5% and 8% fennel oil-containing aerosol and cream produced 84% and 70% repellency, respectively, at 90 min after exposure, whereas Baby Keeper cream and MeiMei cream gave 71% and 57% repellency at 90 min after exposure, respectively, and Repellan S aerosol gave 89% repellency at 210 min. The species and ratio of mosquitoes collected were the genera Culex (44.1%), Anopheles (42.2%), Aedes (7.8%) and Armigeres (5.9%). Fennel oil-containing products could be useful for protection from humans and domestic animals from vector-borne diseases and nuisance caused by mosquitoes.

In order to characterize the T helper 1 (Th1) cytokines of llama, we have cloned several llama cytokine genes and compared them to those of other mammalian species. The cDNAs encoding for interleukin (IL)-2, interferon (IFN), IL-12p35 and IL-12p40 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-2, IFN-, IL-12 p35 and IL-12p40 were found to be 465, 501, 669 or 993 bp in length, with open reading frames encoding 154, 166, 222 or 330 amino acids, respectively. Homology analyses of nucleotide and deduced amino sequences of llama IL-2, IFN-, IL-12p35 and IL-12p40 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla, which includes pig and cattle.

Isolated noncompaction of the ventricular myocardium (INVM) is a rare cardiomyopathy resulting from a failure of normal endomyocardial embryogenesis and it has been categorized as a form of unclassified cardiomyopathy. The disorder is characterized by an excessively prominent trabecular meshwork with deep intertrabecular recesses. Although the disorder is sporadic, familial incidence may occur. Clinical symptoms and prognosis of INVM may differ markedly, and range from an asymptomatic course to a severe cardiac disability. The diagnostic method of choice for IVNM is echocardiography, which reveals multiple prominent trabeculations with deep intertrabecular spaces communicating with the left ventricular cavity in the middle and apical segments of the left ventricle. The authors report a case of INVM in a family in which three adult members (a brother and two sisters) were found to be affected by this disorder. They were all asymptomatic. The diagnosis of the disorder was made first in the 36-year-old brother by transthoracic echocardiography (TTE) and multidetector CT (MD CT), during the process of preoperative evaluation for surgical treatment of low back intervertebral herniated disc. TTE and MD CT showed similar and peculiar findings of INVM. Echocardiographic screening in all first-degree relatives of this patient, in order to identify asymptomatic patients, demonstrated INVM in two elder sisters.

Nucleocapsid protein (NP) gene of Newcastle disease virus (NDV) strains mainly isolated in Japan from 1930 to 2001 was genetically characterized. By deduced amino acid sequence comparison, the N-terminal region (from 1 to 401 residues) of the NP protein was found to be highly conserved, while the C-terminal region was highly variable among the NDV isolates. A phylogenetic tree construct based on the nucleotide sequence of the complete NP gene revealed that the old (prior to 1970s) and the new (after 1980s) isolates could be classified into two major different groups, i.e., a group comprising virulent strains, and another group composed of avirulent strains. By restriction enzyme analysis using Pst I, none of the virulent strains were cleaved, while avirulent strains were cleaved. The results may be useful for simple primary screening test for differentiating NDV isolates.

The promyelocytic leukemia protein (PML) plays an essential role in multiple pathways of apoptosis. Our previous study showed that PML enhances tumor necrosis factor-induced apoptosis by inhibiting the NFkappaB survival pathway. To continue exploring the mechanism of PML-induced apoptosis, we performed a DNA microarray screening of PML target genes using a PML-inducible stable cell line. We found that Survivin was one of the downstream target genes of PML. Cotransfection experiments demonstrated that PML4 repressed transactivation of the Survivin promoter in an isoform-specific manner. Western blot analysis demonstrated that induced PML expression down-regulated Survivin. Inversely, PML knockdown by siRNA up-regulated Survivin expression. A substantial increase in Survivin expression was found in PML-deficient cells. Re-expression of PML in PML-/- mouse embryo fibroblasts down-regulated the expression of Survivin. Furthermore, cells arrested at the G2/M cell cycle phase expressed a high level of Survivin and a significantly lower level of PML. Overexpression of PML in A549 cells reduced Survivin expression leading to massive apoptotic cell death associated with activation of procaspase 9, caspase 3, and caspase 7. Together, our results demonstrate a novel mechanism of PML-induced apoptosis by down-regulation of Survivin.

Yeast is a good system for studying molecular mechanisms of metal tolerance. Using a mini-Tn mutagenized yeast pool, we isolated a chromate-tolerant mutant, CrT9, that displayed metal-specific tolerance since it was only tolerant to Cr(VI), not to Cr(III), Cd, As, or Fe. The Cr-tolerance of CrT9 appeared to be due to reduced Cr accumulation as it accumulated only 56% as much as WT (Y800). Using IPCR (inverse PCR), we found that the mini-Tn had been inserted at nt 741 of the transcriptional activator, MSN1. MSN1 is a multifunctional protein involved in invertase activity, iron uptake, starch degradation, pseudohyphal growth, and osmotic gene expression. We found that there was only one mini-Tn insertion in CrT9 since MSN1 and mini-Tn probes hybridized to the same DNA fragment, and the MSN1 probe detected an enlarged MSN1 mRNA. When we over-expressed MSN1 in CrT9 and WT, both accumulated larger amounts of Cr. We conclude that Cr accumulation in S. cerevisiae is promoted by the transcriptional activator MSN1.

Objectives: To examine the anti-oncogenic effects of promyelocytic leukemia (PML) on bladder cancer and to explore its molecular mechanisms of growth suppression., Methods: Wild-type PML was transfected into bladder cancer cells (5637 cell) and expressed in a replication-deficient adenovirus-mediated gene delivery system and introduced into human bladder cancer cells (5637 cell) in vitro and in vivo. The effect and mechanisms of the PML gene in cell growth, clonogenicity, and tumorigenicity of bladder cancer cells were studied using in vitro and in vivo growth assays, soft agar colony-forming assay, cell cycle analysis, apoptosis assay and in vivo tumorigenicity assay., Results: Overexpression of PML in 5637 cells significantly reduced their growth rate and clonogenicity on soft agar. PML suppressed bladder cancer cell growth by inducing G1 cell cycle arrest and apoptosis. Adenovirus-mediated PML (Ad-PML) significantly suppressed the tumorigenicity and growth of bladder cancer cells. Intratumoral injection of Ad-PML into tumors induced by 5637 cells dramatically suppressed their growth., Conclusions: The results indicated that overexpression of PML protein may promote efficient growth inhibition of human bladder cancer cells by inducing G1 cell cycle arrest and apoptosis, and adenovirus-mediated PML (Ad-PML) expression efficiently suppresses human bladder cancer growth.

The PML tumor suppressor gene is consistently disrupted by t(15;17) in patients with acute promyelocytic leukemia. Promyelocytic leukemia protein (PML) is a multifunctional protein that plays essential roles in cell growth regulation, apoptosis, transcriptional regulation, and genome stability. Our study here shows that PML colocalizes and associates in vivo with the DNA damage response protein TopBP1 in response to ionizing radiation (IR). Both PML and TopBP1 colocalized with the IR-induced bromodeoxyuridine single-stranded DNA foci. PML and TopBP1 also colocalized with Rad50, Brca1, ATM, Rad9, and BLM. IR and interferon (IFN) coinduce the expression levels of both TopBP1 and PML. In PML-deficient NB4 cells, TopBP1 was unable to form IR-induced foci. All-trans-retinoic acid induced reorganization of the PML nuclear body (NB) and reappearance of the IR-induced TopBP1 foci. Inhibition of PML expression by siRNA is associated with a significant decreased in TopBP1 expression. Furthermore, PML-deficient cells express a low level of TopBP1, and its expression cannot be induced by IR or IFN. Adenovirus-mediated overexpression of PML in PML(-/-) mouse embryo fibroblasts substantially increased TopBP1 expression, which colocalized with the PML NBs. These studies demonstrated a mechanism of PML-dependent expression of TopBP1. PML overexpression induced TopBP1 protein but not the mRNA expression. Pulse-chase labeling analysis demonstrated that PML overexpression stabilized the TopBP1 protein, suggesting that PML plays a role in regulating the stability of TopBP1 in response to IR. Together, our findings demonstrate that PML regulates TopBP1 functions by association and stabilization of the protein in response to IR-induced DNA damage.

The promyelocytic leukemia protein (PML) is a growth/tumor suppressor essential for induction of apoptosis by diverse apoptotic stimuli. The mechanism by which PML regulates cell death remains unclear. In this study we found that ectopic expression of PML potentiates cell death by apoptosis in the tumor necrosis factor alpha (TNFalpha)-resistant cell line U2OS and other cell lines. Treatment with TNFalpha significantly sensitized these cells to apoptosis in a p53-independent manner. PML/TNFalpha-induced cell death is associated with DNA fragmentation, activation of caspase-3, -7, and -8, and degradation of DNA fragmentation factor/inhibitor of CAD. PML/TNFalpha-induced cell death could be blocked by the caspase-8 inhibitors CrmA and c-FLIP but not by Bcl-2. These findings indicate that this cell death event is initiated through the death receptor-dependent apoptosis pathway. PML is a transcriptional repressor of NF-kappaB by interacting with RelA/p65 and prevents its binding to the cognate enhancer through the C terminus. Coimmunoprecipitation and double-color immunofluorescence staining demonstrated that PML physically interacts with RelA/p65 in vivo and the two proteins colocalized at the endogenous levels. Overexpression of NF-kappaB rescued cell death induced by PML/TNFalpha. Furthermore, PML(-/-) mouse embryo fibroblasts are more resistant to TNFalpha-induced apoptosis. Together this study defines a novel mechanism by which PML induces apoptosis through repression of the NF-kappaB survival pathway.

Exploration of new ferroic (ferroelectric, ferromagnetic or ferroelastic) materials continues to be a central theme in condensed matter physics and to drive advances in key areas of technology. Here, using thin-film composition spreads, we have mapped the functional phase diagram of the Ni-Mn-Ga system whose Heusler composition Ni(2)MnGa is a well known ferromagnetic shape-memory alloy. A characterization technique that allows detection of martensitic transitions by visual inspection was combined with quantitative magnetization mapping using scanning SQUID (superconducting quantum interference device) microscopy. We find that a large, previously unexplored region outside the Heusler composition contains reversible martensites that are also ferromagnetic. A clear relationship between magnetization and the martensitic transition temperature is observed, revealing a strong thermodynamical coupling between magnetism and martensitic instability across a large fraction of the phase diagram.

Background: Ketamine increases blood pressure and heart rate by unknown mechanisms, but studies suggest that an intact central nervous system and arterial baroreceptors are required. In the brain stem, medial nucleus tractus solitarius receives afferents from nodose neurons that initiate cardiovascular autonomic reflexes. Here, the authors assessed ketamine actions on afferent medial nucleus tractus solitarius synaptic transmission., Methods: Ketamine was applied to horizontally sliced brain stems. Solitary tract (ST) stimulation evoked excitatory postsynaptic currents (eEPSCs) in medial nucleus tractus solitarius neurons. Capsaicin (200 nm) block of ST eEPSCs sorted neurons into sensitive (n = 19) and resistant (n = 23). In nodose ganglion slices, shocks to the peripheral vagal trunk activated afferent action potentials in sensory neurons classified by conduction velocities and capsaicin., Results: Ketamine potently (10-100 mciro m) blocked small, ST-evoked -methyl-d-aspartate synaptic currents found only in a subset of capsaicin-resistant neurons (6 of 12). Surprisingly, ketamine reversibly inhibited ST eEPSC amplitudes and induced synaptic failure at lower concentrations in capsaicin-sensitive than in capsaicin-resistant neurons (P < 0.005; n = 11 and 11). Spontaneous EPSCs using non- -methyl-d-aspartate receptors were insensitive even to 1-3 mm ketamine, suggesting that ST responses were blocked presynaptically. Similarly, ketamine blocked C-type action potential conduction at lower concentrations than A-type nodose sensory neurons., Conclusion: The authors conclude that ketamine inhibits postsynaptic -methyl-d-aspartate receptors and presynaptic afferent processes in medial nucleus tractus solitarius. Unexpectedly, capsaicin-sensitive (C-type), unmyelinated afferents are significantly more susceptible to block than capsaicin-resistant (A-type), myelinated afferents. This differentiation may be related to tetrodotoxin-resistant sodium currents. Since C-type afferents mediate powerful arterial baroreflexes effects, these differential actions may contribute to ketamine-induced cardiovascular dysfunction.

Meq is one of the candidate oncogenes in the MDV1 genome. We previously reported a difference in the meq open reading frame (ORF) between oncogenic and non-oncogenic MDV1: L-meq, in which a 180-bp sequence is inserted into the meq ORF, is detected in non-oncogenic MDV1. To study the functions of a gene product of L-meq (L-MEQ), transactivation by L-MEQ was analyzed by dual luciferase assay using a reporter gene under the control of long (-1--873 bp) and short (-1 - -355 bp) meq promoter (LMP and SMP, respectively). LMP showed higher promoter function than SMP. L-MEQ transactivated the expression of the reporter gene, but less than MEQ did. In the presence of SMP or the cytomegalovirus immediate-early promoter, the same or slightly higher transactivation was observed in cells cotransfected with both meq and L-meq than cells transfected only with meq. However, in the presence of LMP, lower transactivation was observed in cells cotransfected with both meq and L-meq than cells transfected only with meq, suggesting that L-MEQ can be a transrepressor. Replication of a vvMDV1 was enhanced in the cells with meq. Interestingly, however, replication of vvMDV1 was suppressed in the cells with L-meq or with both L-meq and meq, compared to untransfected cells. Thus, L-MEQ could suppress replication of vvMDV1 displaying the meq gene in coinfected cells.

The meq gene encoding a 339-amino-acid bZIP transactivator protein has been identified as a candidate oncogene of Marek's disease virus serotype 1 (MDV1), which induces malignant lymphomas in chickens. We have previously reported that, in addition to meq, L-meq, in which a 180-bp sequence is inserted into the region encoding the transactivation domain of meq, is also detected in chickens experimentally infected with MDV. To further analyze the diversity in meq, PCR was performed using a primer set which specifically amplify the proline-rich repeat (PRR) region in the transactivation domain of meq. In CVI988/R6, a vaccine strain of MDV1, and JM, an MDV1 strain attenuated by prolonged passage in vitro, a major band of a 0.8 kb corresponding to L-meq as well as a minor band of 0.6 kb corresponding to meq was detected by PCR. Furthermore, extra 0.5- and 0.3-kb bands, corresponding to genes termed as short meq (S-meq), and very short meq (VS-meq), respectively, were also detected. These genes were also detected in MDV-transformed cell lines, MSB1 and MTB1. In Md5, an oncogenic MDV1, attenuated by prolonged passage in vitro, the 0.6-kb meq was consistently detected, and 0.5-kb S-meq was occasionally detected. This diversity in meq was due to the difference in the copy number of the PRR region: L-meq and meq contained 9 and 6 copies of PRR while 4 and 2 copies of PRR were present in S-meq and VS-meq, respectively. Thus, the meq gene is polymorphic in the attenuated MDV1 and the MDV-transformed cell lines, and gene products from different meq genes may have different functions from each other.

The repellent activity of materials derived from the methanol extract of fruits from Foeniculum vulgareagainst hungry Aedes aegypti females was examined using skin and patch tests and compared with that of the commercial N,N-diethyl-m-toluamide (deet) and (Z)-9-octadecenoic acid. The biologically active constituents of the Foeniculum fruits were characterized as (+)-fenchone and (E)-9-octadecenoic acid by spectroscopic analyses. Responses varied according to compound, dose, and exposure time. In a skin test with female mosquitoes, at a dose of 0.4 mg/cm(2), (+)-fenchone and (Z)-9-octadecenoic acid exhibited moderate repellent activity at 30 min after treatment, whereas deet provided >1 h of protection against adult mosquitoes at 0.2 mg/cm(2). (Z)-9-Octadecenoic acid was a more potent repellent agent than (E)-9-octadecenoic acid. (+)-Fenchone and (E)-9-octadecenoic acid merit further study as potential mosquito repellent agents or as lead compounds.

Background: Pentobarbital decreases the gain of the baroreceptor reflex on the order of 50%, and this blunting is caused nearly entirely by decreasing cardioinhibitory parasympathetic activity. The most likely site of action of pentobarbital is the gamma-aminobutyric acid type A (GABA(A)) receptor. The authors tested whether pentobarbital augments the inhibitory GABAergic neurotransmission to cardiac parasympathetic neurons, and whether expression of the GABA(A) epsilon subunit prevents this facilitation., Methods: The authors used a novel approach to study the effect of pentobarbital on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with a fluorescent tracer and were visually identified for patch clamp recording. The effects of pentobarbital on spontaneous GABAergic synaptic events were tested. An adenovirus was used to express the epsilon subunit of the GABA(A) receptor in cardiac parasympathetic neurons to examine whether this transfection alters pentobarbital-mediated changes in GABAergic neurotransmission., Results: Pentobarbital increased the duration but not the frequency or amplitude of spontaneous GABAergic currents in cardiac parasympathetic neurons. Transfection of cardiac parasympathetic neurons with the epsilon subunit of the GABA(A) receptor prevented the pentobarbital-evoked facilitation of GABAergic currents., Conclusions: Pentobarbital, at clinically relevant concentrations, prolongs the duration of spontaneous inhibitory postsynaptic currents that impinge on cardiac parasympathetic neurons. This action would augment the inhibition of cardiac parasympathetic neurons, reduce parasympathetic cardioinhibitory activity, and increase heart rate. Expression of the GABA(A) receptor epsilon subunit in cardiac parasympathetic neurons renders the GABA receptors insensitive to pentobarbital.

The promyelocytic leukemia (PML) protein is a tumor suppressor that is disrupted by the chromosomal translocation t(15;17), a consistent cytogenetic feature of acute promyelocytic leukemia. A role of PML in multiple pathways of apoptosis was conclusively demonstrated using PML(-/-) animal and cell culture models. In a previous study, we found that PML sensitizes tumor necrosis factor-induced apoptosis in tumor necrosis factor (TNF)-resistant U2OS cells. This finding helped to explain the mechanism of PML-induced apoptosis. The zinc finger protein A20 is a target gene of NF kappa B inducible by TNF alpha, and it is a potent inhibitor of TNF-induced apoptosis. In the this study, we demonstrated that PML is a transcriptional repressor of the A20 promoter and that PML represses A20 expression induced by TNF alpha. We showed that PML inhibits A20 transactivation through the NF kappa B site by interfering with its binding to the promoter. We also showed that stable overexpression of A20 inhibits apoptosis and caspase activation induced by PML/TNF alpha. The results of this study suggest that A20 is a downstream target of PML-induced apoptosis and supports a role of A20 in modulating cell death induced by PML/TNF alpha in TNF-resistant cells.

In recent years, hybrid neural network approaches, which combine mechanistic and neural network models, have received considerable attention. These approaches are potentially very efficient for obtaining more accurate predictions of process dynamics by combining mechanistic and neural network models in such a way that the neural network model properly accounts for unknown and nonlinear parts of the mechanistic model. In this work, a full-scale coke-plant wastewater treatment process was chosen as a model system. Initially, a process data analysis was performed on the actual operational data by using principal component analysis. Next, a simplified mechanistic model and a neural network model were developed based on the specific process knowledge and the operational data of the coke-plant wastewater treatment process, respectively. Finally, the neural network was incorporated into the mechanistic model in both parallel and serial configurations. Simulation results showed that the parallel hybrid modeling approach achieved much more accurate predictions with good extrapolation properties as compared with the other modeling approaches even in the case of process upset caused by, for example, shock loading of toxic compounds. These results indicate that the parallel hybrid neural modeling approach is a useful tool for accurate and cost-effective modeling of biochemical processes, in the absence of other reasonably accurate process models., (Copyright 2002 Wiley Periodicals, Inc.)

The promyelocytic leukemia protein PML is a tumor and growth suppressor and plays an important role in a multiple pathways of apoptosis and regulation of cell cycle progression. Our previous studies and others also documented a role of PML in transcriptional regulation through its association with transcription coactivator CBP and transcription corepressor HDAC. Here, we showed that PML is a potent transcriptional repressor of Nur77, an orphan receptor and a member of the steroid receptor superfamily of proteins. We found that PML represses Nur77-mediated transactivation through a physical and functional interaction between the two proteins. PML interacts with Nur-77 in vitro in a GST-pull down assay and in vivo by coimmunoprecipitation assay. PML/Nur77 colocalized in vivo in a double immunofluorescent staining and confocal microscopic analysis. Our study further showed that the coiled-coil domain of PML interacts with the DNA-binding domain of Nur77 (amino acids 267-332). Electrophoretic mobility shift assay demonstrated that PML interferes with Nur77 DNA binding in a dose-dependent manner. This study indicates that PML interacts with the DNA-binding domain of Nur77 and represses transcription by preventing it from binding to the target promoter. This study supports a role of PML/Nur77 interaction in regulating cell growth and apoptosis.

Data on the worldwide distribution of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) is limited. A prevalence study of antibodies to BIV and BLV was conducted in six different cattle herds in Brazil. Out of a total of 238 sera analyzed, 11.7% were found positive for anti-BIV p26 antibodies as determined by Western blot analysis, 2.1% were positive for anti-BLV gp51 antibodies as detected by immunodiffusion test. Peripheral blood mononuclear cells from BIV seropositive cattle were found to have BIV-provirus DNA, as detected by nested polymerase chain reaction. A nucleotide sequence corresponding to a 298 bp fragment of the BIV pol gene was also analyzed. Amino acid sequences of these Brazilian pol gene products showed 98.0 to 100% homology to the American strain BIV R29, 97.0 to 99.0% to Japanese BIV isolates, and divergence ranged from 0 to 4.0% among Brazilian BIV isolates. This evidence of the presence of BIV and BLV infections in Brazil should be considered a health risk to Brazilian cattle populations and a potential causative agent of chronic disease in cattle.

In the genome of strains of very virulent Marek's disease virus serotype 1(vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF, termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CV1988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.

Background: Isoflurane inhibits baroreflex control of heart rate (HR) by poorly understood mechanisms. The authors examined whether suprapontine central nervous system cardiovascular regulatory sites are required for anesthetic depression., Methods: The effects of isoflurane (1 and 2 rat minimum alveolar concentration [MAC]) on the baroreflex control of HR were determined in sham intact and midcollicular-transected decerebrate rats. Intravenous phenylephrine (0.2-12 microg/kg) and nitroprusside (1-60 microg/kg) were used to measure HR responses to peak changes in mean arterial pressure (MAP). Sigmoidal logistic curve fits to HR-MAP data assessed baroreflex sensitivity (HR/MAP), HR range, lower and upper HR plateau, and MAP at half the HR range (BP50). Four groups (two brain intact and two decerebrate) were studied before, during, and after isoflurane. To assess sympathetic and vagal contributions to HR baroreflex, beta-adrenoceptor (1 mg/kg atenolol) or muscarinic (0.5 mg/kg methyl atropine) antagonists were administered systemically., Results: Decerebration did not alter resting MAP and HR or baroreflex parameters. Isoflurane depressed baroreflex slope and HR range in brain-intact and decerebrate rats. In both groups, 1 MAC reduced HR range by depressing peak reflex tachycardia. Maximal reflex bradycardia during increases in blood pressure was relatively preserved. Atenolol during 1 MAC did not alter maximum reflex tachycardia. In contrast, atropine during 1 MAC fully blocked reflex bradycardia. Therefore, 1 MAC predominantly depresses sympathetic components of HR baroreflex. Isoflurane at 2 MAC depressed both HR plateaus and decreased BP50 in both groups., Conclusions: Isoflurane depresses HR baroreflex control by actions that do not require suprapontine central nervous system sites. Isoflurane actions seem to inhibit HR baroreflex primarily by the sympathetic nervous system.

Background: Ketamine increases both blood pressure and heart rate, effects commonly thought of as sympathoexcitatory. The authors investigated possible central nervous system actions of ketamine to inhibit cardiac parasympathetic neurons in the brainstem by inhibiting multiple nicotinic excitatory mechanisms., Methods: The authors used a novel in vitro approach to study the effect of ketamine on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with the fluorescent tracer by placing rhodamine into the pericardial sac. Dye-labeled neurons were visually identified for patch clamp recording. The effects of ketamine were tested on nicotine-evoked ligand-gated currents and spontaneous glutamatergic miniature synaptic currents (mini) in cardiac parasympathetic preganglionic neurons., Results: Ketamine (10 microm) inhibited (1) the nicotine (1 microm)-evoked presynaptic facilitation of glutamate release (mini frequency, 18 +/- 7% of control; n = 9), and (2) the direct postsynaptic ligand-gated current (27 +/- 8% of control; n = 9), but ketamine did not alter the amplitude of postsynaptic miniature non-N-methyl-D-aspartate currents. alpha Bungarotoxin, an antagonist of alpha 7 containing nicotinic presynaptic receptors, blocked ketamine actions on mini frequency (n = 10) but not mini amplitude., Conclusions: Ketamine inhibits the presynaptic nicotinic receptors responsible for facilitating neurotransmitter release, as well as the direct ligand-gated inward current, but does not alter the nicotinic augmentation of non-N-methyl-D-aspartate currents in brainstem parasympathetic cardiac neurons. Such actions may mediate the decrease in parasympathetic cardiac activity and increase in heart rate that occurs with ketamine.

Background: Ketamine increases both blood pressure and heart rate, effects commonly thought of as sympathoexcitatory. The authors investigated the possibility that ketamine increases heart rate by inhibiting the central cardiac parasympathetic mechanisms., Methods: We used a novel in vitro approach to study the effect of ketamine on the identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with the fluorescent tracer by placing rhodamine into the pericardial sac. Dye-labeled neurons were visually identified for patch clamp recording, and ketamine effects on isolated potassium (K+) and sodium (Na+) currents were studied., Results: Cardiac nucleus ambiguus neurons (n = 14) were inherently silent, but depolarization evoked sustained action potential trains with little delay or adaptation. Ketamine (10 microm) reduced this response but had no effect on the voltage threshold for action potentials (n = 14; P > 0.05). The current-voltage relations for the transient K+ current and the delayed rectified K+ current (n = 5) were unaltered by ketamine (10 mum-1 mm). Ketamine depressed the total Na+ current dose-dependently (10 microm-1 mm). In addition, ketamine shifted the Na+ current inactivation curves to more negative potentials, thus suggesting the enhancement of the Na+ channel inactivation (P < 0.05; n = 7). In the presence of Cd2+, ketamine (10 mum) continued to inhibit voltage-gated Na+ currents, which recovered completely within 10 min., Conclusions: Ketamine inhibits Na+ but not K+ channel function in brainstem parasympathetic cardiac neurons, and such actions may mediate the decrease in parasympathetic cardiac activity and increase in heart rate that occurs with ketamine.

The insecticidal activities of materials derived from the fruit of star anise, Illicium verum, against adults of Blattella germanica were examined by direct contact application and fumigation methods, and compared with those of DDVP, deltamethrin and hydramethylnon. The biologically active constituent of the Illicium fruit was characterized as the phenylpropene, (E)-anethole, by spectroscopic analysis. In a filter paper diffusion test with females, (E)-anethole caused 80.3% mortality at 0.159 mg cm-2 at 1 and 3 days after treatment (DAT), whereas 16.7% mortality at 3 DAT was achieved at 0.079 mg cm-2. DDVP and deltamethrin gave > 90% mortality at 0.019 mg cm-2 at 1 DAT. At 0.009 mg cm-2, DDVP and deltamethrin showed 73.3 and 60% mortality at 1 DAT, respectively, but 93.3 and 76.7% mortality at 3 DAT. Hydramethylnon exhibited 0 and 93.3% mortality at 0.159 mg cm-2 at 1 and 3 DAT, respectively, whereas 6.7% mortality at 3 DAT was observed at 0.079 mg cm-2. In a fumigation test with females, (E)-anethole was much more effective in closed cups than in open ones, indicating that the insecticidal activity of the compound was largely attributable to fumigant action. (E)-Anethole and DDVP caused 100% mortality at 0.398 and 0.051 mg cm-2 4 and 1 h after treatment, respectively. (E)-Anethole showed 46.7% mortality at 0.199 mg cm-2 at 3 DAT, whereas deltamethrin and hydramethylnon at 0.796 mg cm-2 was ineffective for 3-day period. As naturally occurring insect-control agents, the I verum fruit-derived materials described could be useful for managing populations of B germanica.

To understand the long-term metabolic and functional consequences of increased GLUT4 content, intracellular substrate utilization was investigated in isolated muscles of transgenic mice overexpressing GLUT4 selectively in fast-twitch skeletal muscles. Rates of glycolysis, glycogen synthesis, glucose oxidation, and free fatty acid (FFA) oxidation as well as glycogen content were assessed in isolated EDL (fast-twitch) and soleus (slow-twitch) muscles from female and male MLC-GLUT4 transgenic and control mice. In male MLC-GLUT4 EDL, increased glucose influx predominantly led to increased glycolysis. In contrast, in female MLC-GLUT4 EDL increased glycogen synthesis was observed. In both sexes, GLUT4 overexpression resulted in decreased exogenous FFA oxidation rates. The decreased rate of FFA oxidation in male MLC-GLUT4 EDL was associated with increased lipid content in liver, but not in muscle or at the whole body level. To determine how changes in substrate metabolism and insulin action may influence energy balance in an environment that encouraged physical activity, we measured voluntary training activity, body weight, and food consumption of MLC-GLUT4 and control mice in cages equipped with training wheels. We observed a small decrease in body weight of MLC-GLUT4 mice that was paradoxically accompanied by a 45% increase in food consumption. The results were explained by a marked fourfold increase in voluntary wheel exercise. The changes in substrate metabolism and physical activity in MLC-GLUT4 mice were not associated with dramatic changes in skeletal muscle morphology. Collectively, results of this study demonstrate the feasibility of altering muscle substrate utilization by overexpression of GLUT4. The results also suggest that as a potential treatment for type II diabetes mellitus, increased skeletal muscle GLUT4 expression may provide benefits in addition to improvement of insulin action.

The growth suppressor promyelocytic leukemia protein (PML) is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL). PML plays a key role in multiple pathways of apoptosis and regulates cell cycle progression. The present study demonstrates that PML represses transcription by functionally and physically interacting with histone deacetylase (HDAC). Transcriptional repression mediated by PML can be inhibited by trichostatin A, a specific inhibitor of HDAC. PML coimmunoprecipitates a significant level of HDAC activity in several cell lines. PML is associated with HDAC in vivo and directly interacts with HDAC in vitro. The fusion protein PML-RARalpha encoded by the t(15;17) breakpoint interacts with HDAC poorly. PML interacts with all three isoforms of HDAC through specific domains, and its expression deacetylates histone H3 in vivo. Together, the results of our study show that PML modulates histone deacetylation and that loss of this function in APL alters chromatin remodeling and gene expression. This event may contribute to the development of leukemia.

Patients with normal-karyotype acute myelogenous leukemia (NKAML) may have undetected genetic abnormalities that could affect prognosis. Screening for known AML-specific genetic abnormalities using the reverse transcription polymerase chain reaction (RT-PCR) may help in arriving at a more definitive prognosis. To test this hypothesis, 104 patients without translocation (8;21) and inversion(16), as shown by standard cytogenetic (SC) analysis, were screened for these two genetic abnormalities using RT-PCR. Western blot analysis for the AML1/ETO fusion protein and fluorescent in situ hybridization (FISH) analysis for t(8;21) were performed in patients for whom we had samples. The characteristics and outcome after high-dose cytarabine containing treatments in five patients with t(8;21) shown by RT-PCR alone were then compared to 21 patients with t(8;21) detected using SC analysis. Eight of the 104 patients had masked t(8;21) and none had masked inv(16), as shown by RT-PCR. Five of 54 patients with NKAML had a detectable AML1/ETO fusion RNA transcript. Western blot analysis showed the AML1/ETO fusion protein in four of the seven patients for whom we had samples among the eight with masked t(8;21) shown by RT-PCR. All patients with t(8;21) shown by RT-PCR had negative FISH results. Ninety percent (n=19) of the patients with t(8;21) shown by SC analysis and 40% (n= 2) of the patients with t(8;21) shown by RT-PCR alone achieved a complete remission (P value 0.03). These data suggest that the outcome of NKAML patients with t(8;21) shown by RT-PCR is not equivalent to patients with t(8;21) by SC studies.

Background: Nuclear factor kappaB (NF-kappaB) is a transcription factor that plays important roles in cell proliferation and in immunity against viral infections. NF-kappaB is a dimer of Rel proteins that is sequestered in the cytoplasm as an inactive form through interaction with an inhibitory kappaB (IkappaB) protein. When IkappaB is degraded, the NF-kappaB dimer will enter the nucleus to activate the target genes. Chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection may activate NF-kappaB and, thus, may modulate cell apoptosis and may be associated with oncogenesis. The role of NF-kappaB in hepatocellular carcinoma (HCC) has not yet been explored., Methods: Immunohistochemical staining to search for active nuclear RelA and nuclear IkappaBalpha proteins were done on formalin fixed liver tissues from 65 patients with HCC and from 9 normal control participants. Nuclear extracts of fresh-frozen tumor and nontumor liver tissues from 37 patients with HCC and from 7 normal controls were tested for NF-kappaB-DNA binding activity by electrophoretic mobility shift assay. The RelA and IkappaBalpha protein expressions were studied by Western blot analysis., Results: Nuclear NF-kappaB stainings were significantly more abundant in HBV-infected or HCV-infected tumors as well as nontumor parts of HCC compared with normal controls. Nuclear NF-kappaB DNA binding activity and nuclear RelA protein expression were greater in tumor tissue compared with nontumor tissue, whereas cytosolic IkappaBalphs protein expression was generally greater in nontumor tissue compared with tumor tissue., Conclusions: Constitutive activation of NF-kappaB was found more frequently in tumor tissue compared with nontumor tissue. It is possible that NF-kappaB overexpression accompanied by dysregulation of IkappaBalpha may play a role in the hepatocarcinogenesis of HBV or HCV infection.

Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We characterized a carnation genomic clone, gDcADC8, in which the deduced polypeptide of ADC was 725 amino acids with a molecular mass of 77.7 kDa. The unusually long 5'-UTR that contained a short upstream open reading frame (uORF) of seven amino acids (MQKSLHI) was predicted to form an extensive secondary structure (free energy of approximately -117 kcal mol-1) using the Zuker m-fold algorithm. The result that an ADC antibody detected two bands of 45 and 33 kDa in a petal extract suggested the full length of the 78 kDa polypeptide precursor converted into two polypeptides in the processing reaction. To investigate the role of the transcript leader in translation, in vitro transcription/translation reactions with various constructs of deletion and mutation were performed using wheat germ extract. The ADC transcript leader affected positively downstream translation in both wheatgerm extract and primary transformant overexpressing ADC gene. It was demonstrated that heptapeptide (8.6 kDa) encoded by the ADC uORF was synthesized in vitro. Both uORF peptide, and the synthetic heptapeptide MQKSLHI of the uORF, repressed the translation of downstream ORF. Mutation of the uORF ATG codon alleviated the inhibitory effect. ORF translation was not affected by either a frame-shift mutation in uORF or a random peptide. To our knowledge, this is the first report to provide evidence that a uORF may inhibit the translation of a downstream ORF, not only in cis but also in trans, and that the leader sequence of the ADC gene is important for efficient translation.

A large scale outbreak of hand-foot-and-mouth disease (HFMD) occurred in Taiwan in 1998, in which more than 80 children died of shock syndrome with pulmonary edema/hemorrhage. Enterovirus 71 was implicated as the cause of this outbreak. In order to understand the virological basis responsible for mortality on this scale, nucleotide sequences of VP1 that is important for serotypic specificity, and the 5'-non-coding region (5'-NCR) that is important for replication efficiency, were analyzed comparatively. Phylogenetic analysis of both VP1 and 5'-NCR of nine EV71 isolates derived from specimens of fatal patients and seven isolates derived from uncomplicated HFMD patients showed that all but one isolate fell into genotype B. The one distinct isolate from a case of uncomplicated HFMD belonged to genotype C that was clustered along with one isolate from Taiwan in 1986. Complete sequence analysis of two selected isolates, one from the spinal cord of a fatal case and one from the vesicle fluid of a patient with mild HFMD, confirmed a high degree (97-100%) of identity in nucleotide sequence throughout the entire genome, except focal regions of 3C and 3'-NCR where the nucleotide homology was 90-91%. The identity of the deduced amino acid sequence in the 3C region that encodes viral proteinase dropped further to 86%, a result of missense mutations at the first nucleotide position of many codons.

The hepatitis C virus (HCV) core protein is a multifunctional protein. It may bind to the death domain of tumor necrosis factor receptor 1 (TNFR1) and to the cytoplasmic tail of lymphotoxin-beta receptor, implying that it may be involved in the apoptosis and anti-apoptosis signaling pathways. In vitro studies have been inconclusive regarding its ability to inhibit or enhance TNF-alpha-induced apoptosis. To address this issue, electrophoretic mobility shift assay (EMSA) and immunohistochemical studies were used to show the activation of nuclear factor kappaB (NF-kappaB) in HCV-infected liver tissues and in HCV core-transfected cells. The activation of NF-kappaB was correlated with the apoptosis assays. The results showed that NF-kappaB activation could be shown in HCV-infected livers and HCV core-transfected cells. The data of EMSA correlated with those of immunohistochemical studies, which revealed a higher frequency of NF-kappaB nuclear staining in HCV-infected than in normal livers. NF-kappaB activation conferred resistance to TNF-alpha-induced apoptosis in HCV core-transfected cells. Inhibition of NF-kappaB activation by pyrrolidine dithiocarbamate sensitized them to TNF-alpha-induced apoptosis. These findings suggest that HCV infection may cause anti-apoptosis by activation of NF-kappaB and implicate a mechanism by which HCV may evade the host's immune surveillance leading to viral persistence and possibly to hepatocarcinogenesis.

The purpose of this study is to compare the speed and ease of establishing intraosseous infusion using a standard bone marrow needle (SBMN; $8) and a new screw-tipped intraosseous needle (Sur-Fast; $42). The study is an experimental design. A total of 42 medical students, without prior IO experience, were recruited as study subjects. Subjects were randomized to perform the IO procedures in one of two models: (1) turkey femur or (2) pork ribs. Each subject performed an initial trial using both IO needles without practice (inexperienced) and a second trial using both IO needles after practice (experienced attempt), such that in total, each subject completed four attempts (two with each needle type). IO placement times were measured, and placement difficulty scores were measured using a 10 cm visual analog scale (VAS). The averaged elapsed time to successful IO completion was significantly shorter for the SBMN in the initial "inexperienced" attempt (33 versus 54 seconds, P = .019), but there was no significant difference in the postpractice "experienced" attempt. VAS difficulty scores were lower (easier) for the SBMN for both inexperienced and experienced trials. Success rates were significantly higher for the Sur-Fast needle during the experienced attempt (95% versus 79%, P < .05), but there was no significant difference in success rates during the inexperienced attempt. The Sur-Fast screw-tipped intraosseous needle does not show superiority over the SBMN in this intraosseous model, therefore its higher cost is difficult to justify based on this study.

The promyelocytic leukemia (PML) gene, which encodes a transformation and growth suppressor, was first identified at the chromosomal translocation break point t(15;17) in acute promyelocytic leukemia (PML). To determine if the PML gene might be involved in other neoplasias such as lung cancer, PML expression was analyzed by immunohistochemical staining and in situ hybridization. Considerable PML protein expression in the PML-oncogenic domain (POD) structure was found in adenocarcinomas (ADC) and squamous cell carcinomas (SCC) of the lung, but was almost completely absent in all the small cell lung carcinomas (SCLC) examined. In situ hybridization showed that both mRNA and DNA of PML were present in SCLC and in normal lung, suggesting that the decreased protein expression was due to either a defect in translation or protein instability, rather than the consequence of decreased transcription or gene deletion. Double staining showed that PML expression was inversely correlated with the proliferation marker Ki-67 and positively correlated with levels of apoptotic cells in these tumors. To determine if the precursor cells of SCLC, the neuroendocrine-producing cells, express PML, double labeling was performed with PML and chromogranin A, a bio-marker for neuroendocrine cells. Neuroendocrine cells from normal tissues were found to be PML positive, indicating that the lack of PML protein in SCLC is associated with the tumorigenic phenotype and is not the result of cell-lineage specificity. Thus, the decreased PML expression may play an important role in SCLC development., (Copyright 2000 Wiley-Liss, Inc.)

Background: Coronary spasm during cardiac catheterization is not unusual. The mechanism of spasm remains uncertain, but is considered to be multifactorial. Many researchers believe that coronary spasm that develops during catheterization is partly spontaneous and partly catheter-induced. Because catheter-induced spasm results from mechanical irritation, we tried to find the iatrogenic factors that predispose patients to coronary spasm during coronary angiography., Methods: Retrospectively, we reviewed the records of 7,295 patients who underwent coronary angiography at our hospital from June, 1983 to November, 1997; coronary spasm was documented in 30 patients, who became the study group. We randomly selected 41 patients who had normal coronary arteries as the normal control group. After reviewing cine films of coronary angiography, we compared these two groups for several parameters. These parameters included the length and diameter of the left main coronary artery (LMC), the angle between the LMC and the aorta, the angle between the catheter tip and the LMC, whether the catheter tip came into contact with the vascular wall and whether there was vessel wall bulging, catheter size and catheter/LMC ratio. This angiographic data and the demographic features, including age, sex, history of hypertension, diabetes mellitus, smoking, previous myocardial infarction, family history of coronary artery disease, cholesterol and triglyceride levels and chest pain character (exertional or rest pain) were compared between the study patient group and the control group., Results: The results disclosed that larger catheter size (7.1 +/- 0.6 mm vs 6.4 +/- 0.7 mm, p < 0.001), smaller LMC diameter (4.2 +/- 0.9 mm vs 4.9 +/- 1.0 mm, p = 0.004), larger catheter/LMC ratio (0.07 +/- 0.05 vs 0.05 +/- 0.03, p = 0.022), catheter contact with the vessel wall (27/30 vs 20/41, p < 0.001) and vessel bulging (18/30 vs 5/41, p < 0.001) were related to catheter-induced coronary spasm. We found that the catheter tip coming into contact with the vessel wall, vessel wall bulging and catheter/LMC ratio (odds ratio 8.92 x 10(14)) were statistically significant factors predisposing patients to catheter-induced coronary spasm., Conclusions: Multiple factors contribute to coronary spasm. Of those, mechanical or iatrogenic factors might predispose patients to spasm during coronary catheterization. These facts deserve our attention, because iatrogenically induced spasms may be avoided by meticulously selecting catheters and manipulating them gently.

Straightforward implementation of the exact pairwise nearest neighbor (PNN) algorithm takes O(N3) time, where N is the number of training vectors. This is rather slow in practical situations. Fortunately, much faster implementation can be obtained with rather simple modifications to the basic algorithm. In this paper, we propose a fast O(tauN2) time implementation of the exact PNN, where tau is shown to be significantly smaller than N, We give all necessary data structures and implementation details, and give the time complexity of the algorithm both in the best case and in the worst case. The proposed implementation achieves the results of the exact PNN with the same O(N) memory requirement.

Objectives: We investigated the possibility that the large pulmonary cavity in tuberculosis (TB) lesions might result from imbalances between tumor necrosis factor-alpha (TNF-alpha) and soluble TNF-alpha receptor forms (sTNF-RI and sTNF-RII), and interleukin-beta (IL-1beta) and IL-1 receptor antagonist (IL-1RA) in sites of local inflammation., Patients and Methods: BAL was performed in 32 patients with active pulmonary TB, and the recovered BAL fluid (BALF) was examined for concentrations of TNF-alpha and its soluble receptor forms, IL-1beta, and IL-1RA. Patients were classified into two groups: group 1, patients with a large cavity (> or = 4 cm) on chest radiographs (n = 15); and group 2, patients with a small cavity (< 4 cm; n = 3) or no cavity (n = 14) on chest radiographs., Results: The concentrations of TNF-alpha, IL-1beta, and IL-1RA in BALF were significantly higher in group 1 patients than in group 2 patients before standardization. The difference was also statistically significant for TNF-alpha and IL-1beta after standardization with urea. Furthermore, group 1 patients had significantly higher ratios of TNF-alpha to sTNF-RI and sTNF-RII and IL-1beta to IL-1RA compared with group 2 patients., Conclusions: These findings suggest that the relative abundance of TNF-alpha and IL-1beta associated with imbalances of secretion of soluble TNF-alpha receptor forms and IL-1RA may have caused tissue necrosis leading to cavity formation in patients with active pulmonary TB.

Background: Because exposure to intravenously administered bupivacaine may alter cardiovascular reflexes, the authors examined bupivacaine actions on baroreflex control of heart rate in conscious rats., Methods: Baroreflex sensitivity (pulse interval vs. systolic blood pressure in ms/mmHg) was determined before, and 1.5 and 15.0 min after rapid intravenous administration of bupivacaine (0.5, 1.0, and 2.0 mg/kg) using heart rate changes evoked by intravenously administered phenylephrine or nitroprusside. The actions on the sympathetic and parasympathetic autonomic divisions of the baroreflex were tested in the presence of a muscarinic antagonist methyl atropine and a beta-adrenergic antagonist atenolol., Results: Within seconds of injection of bupivacaine, mean arterial pressure increased and heart rate decreased in a dose-dependent manner. Baroreflex sensitivity was unaltered after administration of 0.5 mg/kg bupivacaine. In addition, 1 mg/kg bupivacaine at 1.5 min depressed phenylephrine-evoked reflex bradycardia (0.776 +/- 0.325 vs. 0.543 +/- 0.282 ms/mmHg, P < 0.05) but had no effect on nitroprusside-induced tachycardia. Bupivacaine (2 mg/kg), however, depressed reflex bradycardia and tachycardia (phenylephrine, 0.751 +/- 0.318 vs. 0.451 +/- 0.265; nitroprusside, 0.839 +/- 0.256 vs. 0.564 +/- 0.19 ms/mmHg, P < 0.05). Baroreflex sensitivity returned to prebupivacaine levels by 15 min. Bupivacaine (2 mg/kg), in the presence of atenolol, depressed baroreflex sensitivity (phenylephrine, 0.633 +/- 0.204 vs. 0.277 +/- 0.282; nitroprusside, 0.653 +/- 0.142 vs. 0.320 +/- 0.299 ms/mmHg, P < 0.05). In contrast, bupivacaine did not alter baroreflex sensitivity in the presence of methyl atropine., Conclusions: Bupivacaine, in clinically relevant concentrations, inhibits baroreflex control of heart rate in conscious rats. This inhibition appears to involve primarily vagal components of the baroreflex-heart rate pathways.

Accumulating evidence suggests that patients with active pulmonary tuberculosis (TB) have an alveolar inflammation resulting in the release of tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta in bronchoalveolar epithelial fluid. It was proposed that the levels of these cytokines would correlate with clinical status parameters (extent of pulmonary involvement, fever, and body weight loss) and that their naturally occurring inhibitors would be concomitantly released in the local inflammatory sites. To test this hypothesis lung epithelial lining fluid (ELF) obtained by bronchoalveolar lavage and serum were collected from 29 patients with active pulmonary TB and 15 healthy subjects to determine the levels of these variables using a sandwich enzyme-linked immunosorbent assay (ELISA). ELF levels of TNF-alpha, soluble (s)TNF receptor I (RI), sTNF-receptor II (RII) and interleukin-1 receptor antagonist (IL-1RA) but not IL-1beta, and their serum levels except for sTNF-RII and IL-1beta were significantly higher in TB patients. Nevertheless, only ELF levels of TNF-alpha and IL-1beta were significantly correlated with disease status. No correlation was found between TNF-alpha levels and those of sTNF-RI and sTNF-RII, nor between IL-1beta and IL-1RA in ELF and serum of TB patients, although there was a significant correlation between sTNF-RI and sTNF-RII levels both in ELF and serum. These findings suggest local release of tumour necrosis factor-alpha and interleukin-1beta and a correlation with disease status. Soluble tumour necrosis factor-alpha receptors and interleukin-1beta receptor antagonist, although increased in lung epithelial lining fluid and serum in tuberculosis patients, were not correlated with tumour necrosis factor-alpha and interleukin-1beta or with disease status.

AML2 is a member of the acute myelogenous leukemia, AML family of transcription factors. The biologic functions of AML1 and AML3 have been well characterized; however, the functional role of AML2 remains unknown. In this study, we found that AML2 protein expressed predominantly in cells of hematopoietic origin is a nuclear serine phosphoprotein associated with the nuclear matrix, and its expression is not cell cycle-related. In HL-60 cells AML2 expression can be induced by all three natural retinoids, all-trans-retinoic acid (RA), 13-cis-RA, and 9-cis-RA in a dose-dependent manner. A synthetic retinoic acid derivative, 4HPR, which neither activates RA receptor (RAR) alpha nor retinoic X receptor alpha was unable to induce the expression of AML2. A RAR-selective activator, TTNPB, induced AML2 expression similar to RA. Our study further showed that AGN193109, a potent RARalpha antagonist, suppressed AML2 expression induced by RA and that a retinoic X receptor pan agonist AGN194204 had no effect on its expression. Taken together, these studies conclusively demonstrated that the expression of AML2 in HL-60 cells is regulated through the RARalpha-specific signaling pathway. Our study further showed that after all-trans-retinoic acid priming, AML2 expression could be augmented by vitamin D(3). Based on these studies we hypothesize that AML2 expression is normally regulated by retinoid/vitamin D nuclear receptors mainly through the RARalpha-dependent signaling pathway and that it may play a role in hematopoietic cell differentiation.

Abciximab, a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor, reduces ischemic complications of coronary interventions after first administration. In this study, We sought to determine whether readministration of abciximab is associated with equal efficacy and safety. We retrospectively reviewed the charts of 35 patients who received two doses of abciximab at separate intervals. We monitored patients clinically for recurrent ischemia, bleeding complications, and thrombocytopenia. We measured hemoglobin and platelet counts before and after readministration of abciximab. There was no cardiac-related death, myocardial infarction, or recurrent ischemia. No obvious bleeding occurred in any of the 35 patients, although 1 patient had a drop of hemoglobin >3 gm/dl. We observed one episode of severe thrombocytopenia without any complication, and this patient improved without requiring platelet transfusion. There was no profound thrombocytopenia. We conclude that readministration of abciximab was well tolerated without any evidence of altered efficacy or safety. Cathet. Cardiovasc. Intervent. 47:294-296, 1999., (Copyright 1999 Wiley-Liss, Inc.)

Enterovirus 71 (EV71), one of the major causative agents for hand, foot and mouth disease (HFMD), is sometimes associated with severe central nervous system diseases. In 1997, in Malaysia and Japan, and in 1998 in Taiwan, there were HFMD epidemics involving sudden deaths among young children, and EV71 was isolated from the HFMD patients, including the fatal cases. The nucleotide sequences of each EV71 isolate were determined and compared by phylogenetical analysis. EV71 strains from previously reported epidemics belonged to genotype A-1, while those from recent epidemics could be divided into two genotypes, A-2 and B. In Malaysia, genotype A-2 was more prevalent, while in Japan and Taiwan, B genotype was more prevalent. Two isolates from fatal cases in Malaysia and one isolate from a fatal case in Japan were genotype A-2. However, all isolates from three fatal cases in Taiwan belonged to genotype B. The severity of the HFMD did not link directly to certain genotypes of EV71.

Setting: We hypothesized that patients with active pulmonary tuberculosis (TB) have tubercular pneumonitis and that alveolar macrophages at these sites release proinflammatory cytokines, resulting in high levels of cytokines in alveolar epithelial lining fluid., Objective: To measure cytokine levels in bronchoalveolar lavage fluid (BALF) and to confirm the source of any cytokines by examination of alveolar macrophage cytokine mRNA., Design: Seventeen active pulmonary TB patients and 15 healthy controls were prospectively studied. Bronchoalveolar lavage (BAL) was performed, proinflammatory cytokine levels were determined and alveolar macrophages isolated from BALF were prepared for RNA extraction and Northern blot analysis., Results: Compared with healthy controls, TNF-alpha, IL-1 beta and IL-6 in BALF were all significantly higher in patients with active pulmonary TB, 298.7 +/- 85.9 vs. 8.9 +/- 2.7 (P = 0.0001); 164.4 +/- 67.5 vs. 8.9 +/- 2.7 (P = 0.003); 969.2 +/- 214.2 vs. 86.4 +/- 17.0 (mean +/- SE pg/ml) (P = 0.0001), respectively. Only TNF-alpha and IL-6 levels were significantly higher in sera of active pulmonary TB patients, 92.3 +/- 28.7 vs. 3.5 +/- 1.2; 15.2 +/- 5.4 vs. 2.1 +/- 2.1, respectively. Northern blot analysis revealed increased gene expression of these alveolar macrophage cytokines in patients with active pulmonary TB compared healthy controls., Conclusion: Significantly higher levels of TNF-alpha, IL-1 beta and IL-6 were found in BALF from patients with active pulmonary TB, and were released by alveolar macrophages in the TB lesions.