Apoptotic effect of dibenzylideneacetone on oral cancer cells via modulation of specificity protein 1 and Bax.

OBJECTIVE: Dibenzylideneacetone (DBA), an analogue of curcumin, has been shown to have potential anticancer effects against several cancers. However, the molecular mechanism underlying anticancer activity of DBA has not been well established yet. In this study, we investigated the function and molecular mechanism of DBA in human oral cancer cells. MATERIALS AND METHODS: The growth-inhibitory and apoptotic effects and related signaling pathways of DBA were evaluated using trypan blue exclusion assay, 4'-6-diamidino-2-phenylindole staining, Western blot analysis, siRNA, and reverse transcription-polymerase chain reaction. RESULTS: DBA inhibited cell growth and induced apoptosis, as evidenced by PARP cleavage, activation of caspase-3, and nuclear condensation. DBA also decreased specificity protein 1 (Sp1) expression through facilitating protein degradation. In addition, DBA enhanced the induction of pro-apoptotic protein Bax, resulting in their conformational change, translocation into mitochondrial outer membrane, and its oligomerization. The down-regulation of Sp1 by siRNA targeting Sp1 and mithramycin A increasingly activated Bax to trigger apoptosis. Moreover, DBA-induced growth inhibition and apoptosis in various human oral cancer cell lines were associated with Sp1 down-regulation and induction of Bax. CONCLUSION: These findings suggest that DBA may be a potential anticancer drug candidate to induce apoptosis through down-regulation of Sp1 in human oral cancer.

Investigation of MC1R SNPs and Their Relationships with Plumage Colors in Korean Native Chicken.

The melanocortin 1 receptor (MC1R) gene is related to the plumage color variations in chicken. Initially, the MC1R gene from 30 individuals was sequenced and nine polymorphisms were obtained. Of these, three and six single nucleotide polymorphisms (SNPs) were confirmed as synonymous and nonsynonymous mutations, respectively. Among these, three selected SNPs were genotyped using the restriction fragment length polymorphism (RFLP) method in 150 individuals from five chicken breeds, which identified the plumage color responding alleles. The neighbor-joining phylogenetic tree using MC1R gene sequences indicated three well-differentiated different plumage pigmentations (eumelanin, pheomelanin and albino). Also, the genotype analyses indicated that the TT, AA and GG genotypes corresponded to the eumelanin, pheomelanin and albino plumage pigmentations at nucleotide positions 69, 376 and 427, respectively. In contrast, high allele frequencies with T, A and G alleles corresponded to black, red/yellow and white plumage color in 69, 376 and 427 nucleotide positions, respectively. Also, amino acids changes at position Asn23Asn, Val126Ile and Thr143Ala were observed in melanin synthesis with identified possible alleles, respectively. In addition, high haplotype frequencies in TGA, CGG and CAA haplotypes were well discriminated based on the plumage pigmentation in chicken breeds. The results obtained in this study can be used for designing proper breeding and conservation strategies for the Korean native chicken breeds, as well as for the developing breed identification markers in chicken.

Phylogenetic Analysis of a Privately-owned Korean Native Chicken Population Using mtDNA D-loop Variations.

The use of Korean native chicken is increasing, and the discovery of new genetic resources is very important from both economic and genetic conservation points of view. In this study, mtDNA D-loop sequences from 272 privately-owned Korean native chickens from a Hyunin farm were investigated. Seventeen nucleotide substitutions were identified from the sequence analysis and they were classified as 6 haplotypes. Previously investigated haplotypes in five Korean native chicken populations have been compared with the Hyunin chicken population. The results indicated that two haplotypes, H10 and H15, in the Hyunin chicken population were not previously identified in other Korean native chicken populations, representing 33.09% (90/272) and 1.1% (3/272) of the Hyunin population, respectively. On the other hand, four other haplotypes were identical to those of a previous study of Korean native chicken populations. This result is indicative of conservation strategies of Hyunin chicken populations for expanding the genetic diversity in the Korean native chicken population.

Discrimination of korean native chicken lines using fifteen selected microsatellite markers.

In order to evaluate the genetic diversity and discrimination among five Korean native chicken lines, a total of 86 individuals were genotyped using 150 microsatellite (MS) markers, and 15 highly polymorphic MS markers were selected. Based on the highest value of the number of alleles, the expected heterozygosity (He) and polymorphic information content (PIC) for the selected markers ranged from 6 to 12, 0.466 to 0.852, 0.709 to 0.882 and 0.648 to 0.865, respectively. Using these markers, the calculated genetic distance (Fst), the heterozygote deficit among chicken lines (Fit) and the heterozygote deficit within chicken line (Fis) values ranged from 0.0309 to 0.2473, 0.0013 to 0.4513 and -0.1002 to 0.271, respectively. The expected probability of identity values in random individuals (PI), random half-sib (PI half-sibs ) and random sibs (PI sibs ) were estimated at 7.98×10(-29), 2.88×10(-20) and 1.25×10(-08), respectively, indicating that these markers can be used for traceability systems in Korean native chickens. The unrooted phylogenetic neighbor-joining (NJ) tree was constructed using 15 MS markers that clearly differentiated among the five native chicken lines. Also, the structure was estimated by the individual clustering with the K value of 5. The selected 15 MS markers were found to be useful for the conservation, breeding plan, and traceability system in Korean native chickens.

Forming mechanism of the bipolar resistance switching in double-layer memristive nanodevices.

To initiate resistance switching phenomena, it is usually necessary to apply a strong electric field to a sample. This forming process poses very serious obstacles in real nanodevice applications. In unipolar resistance switching (URS), it is well known that the forming originates from soft dielectric breakdown. However, the forming in bipolar resistance switching (BRS) is poorly understood. In this study, we investigated the forming processes in Pt/Ta2O5/TaOx/Pt and Pt/TaOx/Pt nanodevices, which showed BRS and URS, respectively. By comparing the double- and single-layer systems, we were able to observe differences in the BRS and URS forming processes. Using computer simulations based on an 'interface-modified random circuit breaker network model', we could explain most of our experimental observations. This success suggests that the BRS forming in our Pt/Ta2O5/TaOx/Pt double-layer system can occur via two processes, i.e., polarity-dependent resistance switching in the Ta2O5 layer and soft dielectric breakdown in the TaOx layer. This forming mechanism can be used to improve the performance of BRS devices. For example, we could improve the endurance properties of Pt/Ta2O5/TaOx/Pt cells by using a small forming voltage.

Scaling theory for unipolar resistance switching.

We investigate a reversible percolation system showing unipolar resistance switching in which percolating paths are created and broken alternately by the application of an electric bias. Owing to the dynamical changes in the percolating paths, different from those in classical percolating paths, a detailed understanding of the structure is demanding and challenging. Here, we develop a scaling theory that can explain the transport properties of these conducting paths; the theory is based on the fractal geometry of a percolating cluster. This theory predicts that two scaling behaviors emerge, depending on the topologies of the conducting paths. We confirm these theoretical predictions experimentally by observing material-independent universal scaling behaviors in unipolar resistance switching.

An investigation of the impact of the location and timing of antigen-specific T cell division on airways inflammation.

It is widely accepted that allergic asthma is orchestrated by T helper type 2 lymphocytes specific for inhaled allergen. However, it remains unclear where and when T cell activation and division occurs after allergen challenge, and whether these factors have a significant impact on airways inflammation. We therefore employed a CD4-T cell receptor transgenic adoptive transfer model in conjunction with laser scanning cytometry to characterize the location and timing of T cell division in asthma in vivo. Thus, for the first time we have directly assessed the division of antigen-specific T cells in situ. We found that accumulation of divided antigen-specific T cells in the lungs appeared to occur in two waves. The first very early wave was apparent before dividing T cells could be detected in the lymph node (LN) and coincided with neutrophil influx. The second wave of divided T cells accumulating in lung followed the appearance of these cells in LN and coincided with peak eosinophilia. Furthermore, accumulation of antigen-specific T cells in the draining LN and lung tissue, together with accompanying pathology, was reduced by intervention with the sphingosine 1-phosphate receptor agonist FTY720 2 days after challenge. These findings provide greater insight into the timing and location of antigen-specific T cell division in airways inflammation, indicate that distinct phases and locations of antigen presentation may be associated with different aspects of pathology and that therapeutics targeted against leukocyte migration may be useful in these conditions.

Tumour necrosis factor-alpha blockade suppresses murine allergic airways inflammation.

Asthma is a heterogeneous disease that has been increasing in incidence throughout western societies and cytokines, including proinflammatory tumour necrosis factor alpha (TNF-alpha), have been implicated in the pathogenesis of asthma. Anti-TNF-alpha therapies have been established successfully in the clinic for diseases such as rheumatoid arthritis and Crohn's disease. TNF-alpha-blocking strategies are now being trialled in asthma; however, their mode of action is poorly understood. Based on the observation that TNF-alpha induces lymph node hypertrophy we have attempted to investigate this as a mechanism of action of TNF-alpha in airway inflammation by employing two models of murine airway inflammation, that we have termed short and long models, representing severe and mild/moderate asthma, respectively. The models differ by their immunization schedules. In the short model, characterized by eosinophilic and neutrophilic airway inflammation the effect of TNF-alpha blockade was a reduction in draining lymph node (DLN) hypertrophy, eosinophilia, interleukin (IL)-5 production and immunoglobulin E (IgE) production. In the long model, characterized by eosinophilic inflammation, TNF-alpha blockade produced a reduction in DLN hypertrophy and IL-5 production but had limited effects on eosinophilia and IgE production. These results indicate that anti-TNF-alpha can suppress DLN hypertrophy and decrease airway inflammation. Further investigations showed that anti-TNF-alpha-induced inhibition of DLN hypertrophy cannot be explained by preventing l-selectin-dependent capture of lymphocytes into the DLN. Given that overall TNF blockade was able to suppress the short model (severe) more effectively than the long model (mild/moderate), the results suggest that TNF-alpha blocking therapies may be more effective in the treatment of severe asthma.

The control of cell adhesion and viability by zinc oxide nanorods.

The ability to control the behavior of cells that interact with implanted biomaterials is desirable for the success of implanted devices such as biosensors or drug delivery devices. There is a need to develop materials that can limit the adhesion and viability of cells on implanted biomaterials. In this study, we investigated the use of zinc oxide (ZnO) nanorods for modulating the adhesion and viability of NIH 3T3 fibroblasts, umbilical vein endothelial cells, and capillary endothelial cells. Cells adhered far less to ZnO nanorods than the corresponding ZnO flat substrate. The few cells that adhered to ZnO nanorods were rounded and not viable compared to the flat ZnO substrate. Cells were unable to assemble focal adhesions and stress fibers on nanorods. Scanning electron microscopy indicated that cells were not able to assemble lamellipodia on nanorods. Time-lapse imaging revealed that cells that initially adhered to nanorods were not able to spread. This suggests that it is the lack of initial spreading, rather than long-term exposure to ZnO that causes cell death. We conclude that ZnO nanorods are potentially useful as an adhesion-resistant biomaterial capable of reducing viability in anchorage-dependent cells.

Does microbleed predict haemorrhagic transformation after acute atherothrombotic or cardioembolic stroke?

OBJECTIVES: Cerebral microbleeds (MBs) are known to be indicative of bleeding-prone microangiopathy and may predict incident intracerebral haemorrhage. However, there is controversy concerning the causal relationship between the presence of MBs and haemorrhagic transformation (HTf) after ischaemic stroke. METHODS: Of the 1034 patients with acute ischaemic stroke who were consecutively admitted to our hospital, 377 patients with stroke due to large-artery atherothrombosis or cardioembolism were selected for participation in this study. We examined the MBs using T2*-weighted gradient-echo MRI performed within 24 hours after admission, and the incidence of HTf was assessed using follow-up brain MRI during the hospitalisation period. RESULTS: Of the 377 patients with stroke, 234 were male (62.1%) and the mean age was 66.2 +/-11.7 years. MBs were initially found in 109 patients (28.9%), and newly incident HTf was noted during the hospitalisation period in 74 patients (19.6%). The presence of MBs was not increased in the patients with HTf (24.3% vs. 30.0% in the patients without HTf; p = 0.331). In addition, the number of MBs was not higher in the patients with HTf (0.7+/-1.5 vs. 1.8+/-8.1; p = 0.234). This lack of significance between MBs and HTf persisted after stratification by stroke mechanism. CONCLUSIONS: This study suggests that underlying MBs do not predict incident HTf after acute ischaemic stroke. The clinical significance of MBs should be differentially evaluated according to the type of disease (intracerebral haemorrhage vs. HTf).

GaN, ZnO and InN nanowires and devices.

A brief review is given of recent developments in wide bandgap semiconductor nanowire synthesis and devices fabricated on these nanostructures. There is strong interest in these devices for applications in UV detection, gas sensors and transparent electronics.

Activation-dependent degradation of protein kinase C eta.

Prolonged activation of protein kinase Cs (PKCs) by long-term treatment of cells with phorbol ester tumor promoters down-regulates the expression of many PKCs. To investigate the molecular mechanisms involved in the down-regulation of PKC eta, we expressed the novel PKCs eta and θ and various mutant forms in baby hamster kidney cells. Upon overexpression, constitutively active PKC eta, but not wild type or kinase-dead PKC eta, underwent rapid degradation to generate several lower molecular weight polypeptides. When co-expressed with active kinases, kinase-dead PKC eta with a pseudosubstrate site mutation designed to give an active conformation was down-regulated while the wild type PKC eta was not. These results suggest requirements for kinase activity and an active conformation for down-regulation of PKC eta. Treatment with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal and lactacystin led to accumulation of PKC eta proteolytic products and potentially ubiquitinated forms. While wild type PKC eta localizes mostly to the detergent-soluble fraction of the cell, a significant portion of full-length constitutively active PKC eta and of kinase-dead, active conformation PKC eta were found in the detergent-insoluble fraction. Several proteolytic fragments of constitutively active PKC eta also were found in the detergent insoluble fraction. These full-length and proteolytic fragments of PKC eta in the detergent-insoluble fraction accumulated further in the presence of proteasome inhibitors. These data suggest that active conformation PKC eta accumulates in the detergent-insoluble compartment, is degraded by proteolysis in the presence of kinase activity, and that the cleavage products undergo further degradation via ubiquitin-mediated degradation in the proteasome. Oncogene (2000) 19, 4263 - 4272

Pathogenesis of virus-induced immune-mediated demyelination.

Theiler's murine encephalomyelitis virus-induced demyelinating disease has been extensively studied as an attractive infectious model for human multiple sclerosis. Virus-specific inflammatory Th1 cell responses followed by autoimmune responses to myelin antigens play a crucial role in the pathogenic processes leading to demyelination. Antibody and cytotoxic T cells (CTL) responses to virus appears to be primarily protective from demyelinating disease. Although the role of Th1 and CTL responses in the induction of demyelinating disease is controversial, assessment of cytokines produced locally in the central nervous system (CNS) during the course of disease and the effects of altered inflammatory cytokine levels strongly support the importance of Th1 responses in this virus-induced demyelinating disease. Induction of various chemokines and cytokines in different glial and antigen presenting cells upon viral infection appears to be an important initiation mechanism for inflammatory Th1 responses in the CNS. Coupled with the initial inflammatory responses, viral persistence in the CNS may be a critical factor for sustaining inflammatory responses and consequent immune-mediated demyelinating disease.

Enhanced susceptibility to Theiler's virus-induced demyelinating disease in perforin-deficient mice.

Theiler's virus induces immune-mediated demyelinating disease similar to human MS in susceptible mice. Though the MHC class II-restricted T cell response is critical, susceptibility/resistance is also associated with a MHC class I haplotype. Here we report that perforin-deficient C57BL/6 mice (pKO) are susceptible to demyelination and develop clinical disease. The levels of primary demyelination, proliferation, Th1 responses, and viral load were also markedly enhanced. In addition, immunization of pKO mice with UV-inactivated virus further enhanced clinical incidence and accelerated the disease course. Thus, perforin is most likely involved in viral clearance, hence protection from the disease.

Inhibitory effects of resveratrol analogs on unopsonized zymosan-induced oxygen radical production.

Resveratrol, a natural hydroxystilbene, has been reported to have anti-inflammatory and anticarcinogenic activities. Inhibitory effects of resveratrol and its analogs on reactive oxygen species (ROS) production in unopsonized zymosan-stimulated murine macrophage Raw264.7 cells, human monocytes, and neutrophils were analyzed to investigate if the anti-inflammatory and anticarcinogenic activities of resveratrol are related to the inhibition of ROS production. Resveratrol was a potent inhibitor of ROS production in both unopsonized zymosan-stimulated Raw264.7 cells and human monocytes and neutrophils. Resveratrol exhibited 50% inhibition values (IC50) of 17 microM in activated Raw264.7 cells, 18 microM in human monocytes, and 23 microM in human neutrophils. 3,5-Dihydroxy-4'-methoxystilbene or 3,4'-dimethoxy-5-hydroxystilbene exhibited IC50 values of 63 or 73 microM in Raw264.7 cells, 51 or >100 microM in human monocytes, and 10 or 37 microM in human neutrophils, respectively. Trimethylresveratrol, piceid, and 3,5-dihydroxy-4'-methoxystilbene-3-O-beta-D-glucoside were weak inhibitors of ROS production. Thus, resveratrol was identified as a potent inhibitor of ROS production, which might be one biochemical mechanism related to its anti-inflammatory and anticarcinogenic activities. The number and position of hydroxy substituents in resveratrol analogs seem to play an important role in the inhibitory potency of ROS production.

Sprouting of sympathetic nerve fibers into the dorsal root ganglion following peripheral nerve injury depends on the injury site.

Peripheral nerve injury often induces sympathetic nerve fiber sprouting in the dorsal root ganglion (DRG) and injured nerve. Presently, the underlying mechanism and functional significance of the sprouting are unknown. This study was performed to see whether the degree of the sprouting in the DRG was a function of the distance between the DRG and injury site. To this aim, we compared two groups of rats with respect to the sympathetic nerve fibers sprouting in the S1-3 DRG; one group was subjected to unilateral inferior and superior caudal trunk transections at the level between the S3 and S4 spinal nerves (S34 group) and the other group at the levels between the S1 and S2, between S2 and S3 and between S3 and S4 spinal nerves (S123 group). The transections in both groups equally eliminated the inputs from the tail to the S1-3 DRG, but the distance from the S1/S2 DRG to the injury site was different between the two groups. Immunohistochemical staining with tyrosine hydroxylase (TH) antibody of the S1-3 DRG removed from rats a week after the injury revealed that the degree of penetration of TH-positive fibers into the S1 and S2 DRG was much more extensive in the S123 group than in the S34 group, whereas that into the S3 DRG was not significantly different between the two groups. These results suggest that the extent of the sympathetic nerve fiber sprouting in the DRG following peripheral nerve injury is inversely related to the distance between the DRG and injury site.

NMDA recepter-mediated neuroprotection by essential oils from the rhizomes of Acorus gramineus.

Acori graminei Rhizoma (AGR) is shown to exhibit a number of pharmacological actions including sedation and anticonvulsive action. To further characterize its actions in the CNS, the present study evaluated the effects of essential oils (EO) from AGR on the excitotoxic neuronal cell death induced in primary rat cortical cell cultures. EO inhibited the glutamate-induced excitotoxicity in a concentration-dependent manner, with the IC50 of 0.241 mg/ml. EO exerted more potent neuroprotection against the toxicity induced by NMDA (IC50 = 0.139 mg/ml). In contrast, the AMPA-induced toxicity was not inhibited by EO. Receptor-ligand binding studies were performed to investigate the neuroprotective action mechanism. EO dramatically inhibited the specific bindings of a use-dependent NMDA receptorion channel blocker [3H]MK-801, indicating an NMDA receptor antagonist-like action. However, the bindings of [3H]MDL 105,519, a ligand selective for the glycine binding site of NMDA receptor, were not considerably inhibited. These results demonstrated that EO extracted from AGR exhibited neuroprotective effects on cultured cortical neurons through the blockade of NMDA receptor activity, and that the glycine binding site appeared not to be the major site of action.

Hypoxic contraction of isolated rat pulmonary artery.

The effects of hypoxia in precontracted rat pulmonary artery rings with and without endothelium was studied. Hypoxia (30 mmHg) produced a contraction (hypoxic pulmonary vasoconstriction, HPV). Removal of endothelium abolished the HPV. The HPV was reproducible. The amplitude of HPV was similar to arteries equilibrated with 100% O2 and room air. L-NNA markedly inhibited the HPV whereas indomethacin was ineffective. HPV was inhibited by caffeine, but not inhibited by nifedipine. It would be concluded that HPV in isolated rat pulmonary arteries is dependent on the endothelium and the mechanisms involved may be inhibition of basal NO production.

Therapeutic dilemmas in the management of thyroid cancer with laryngotracheal involvement.

Invasion of the larynx and trachea by thyroid cancer is an uncommon but difficult problem. There is no consensus on indication for or extent of surgery, particularly when there is a requirement for airway reconstruction. From 1989 through 1996, we treated 22 patients with thyroid carcinoma with invasion of the larynx and trachea. Seventeen of these patients had recurrent disease. We applied radioactive iodine therapy after regional ablative surgery to resectable tumors with or without lung metastasis, larynx-preserving surgery to extraluminal or small intraluminal tumors restricted to the short segment of trachea, or total laryngectomy to recurrent tumors deeply invading the cartilage framework of the larynx. We performed arytenoid adduction or thyroplasty in one stage if the recurrent laryngeal nerve was paralyzed or resected intraoperatively. We could get relatively good survival and functional results by aggressive surgical treatment in 20 patients, but the disease was inoperable in 2 patients. It is stressed that head and neck surgeons who have to deal with cancer of the thyroid should not only be familiar with various techniques of airway reconstruction and voice rehabilitation but also must be aware of the biologic behavior of the thyroid carcinoma.

Inhibition of excitotoxic neuronal death by methanol extract of Acori graminei rhizoma in cultured rat cortical neurons.

Acori graminei rhizoma (AGR) are reported to exhibit a number of pharmacological actions in the central nervous system. The effects of the methanol extract of AGR on excitotoxic neuronal death were evaluated in the present study using cultured rat cortical neurons. Based on the phase-contrast microscopic examinations of cultures and lactate dehydrogenase activities measured in the culture media, the glutamate-induced excitotoxicity was significantly inhibited by the extract. The inhibitory action of the extract was more potent and selective for the N-methyl-D-aspartate (NMDA) receptor-mediated toxicity. The AGR extract competed with [3H]MDL 105,519 for the specific binding to the glycine site of the NMDA receptor with the IC(50) value of 164.7 microg/ml. Modulation of the NMDA receptor activity by the extract was determined using [3H]MK-801 binding studies. The reduction of the binding in the presence of the extract indicated the receptor inactivation by AGR. These results demonstrated that the methanol extract of AGR exhibited protective action against excitotoxic neuronal death, and that the neuroprotective action was primarily due to the blockade of NMDA receptor function by the interaction with the glycine binding site of the receptor.