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Background: The main strategy against nasopharyngeal carcinoma (NPC) is radiotherapy. However, radioresistance mediated recurrence is a leading clinical bottleneck in NPC. Revealing the mechanism of NPC radioresistance will help improve the therapeutic effect. Methods: In this study, the role of TRIM21 (tripartite motif–containing 21) in NPC receiving ionizing radiation was firstly examined both in vivo and in vitro. Mass spectrometry analysis was performed to identify the downstream targets of TRIM21. NPC cells with TRIM21 or SERPINB5 (serpin family B member 5) overexpression or knockout were used to determine the epistatic relationship among SERPINB5, GMPS (guanine monophosphate synthase) and TRIM21. Flow cytometry, co-immunoprecipitation, western blot and immunofluorescence were employed to strengthen the results. Finally, immunohistochemistry using 4 radiosensitive and 8 radioresistent NPC patient samples was perform to examine the association between SERPINB5 or GMPS expression and patient radio-sensitivity. Results: As an E3 ligase, TRIM21 was highly expressed in NPC. After ionizing radiation, TRIM21 repressed TP53 expression by mediating GMPS ubiquitination and degradation. Overexpression of TRIM21 protected NPC cells from radiation mediated cell apoptosis in vitro and in vivo. Further analysis revealed that TRIM21 mediated GMPS repression was dependent on SERPINB5, and SERPINB5 served as an adaptor which prevented GMPS from entering into the nucleus and introduced TRIM21 for GMPS ubiquitination. Moreover, the in vitro and in vivo results validated the finding that SERPINB5 promoted NPC cell radioresistance, and the radioresistant patients had higher SERPINB5 expression. Conclusions: Overall, our data showed that TRIM21–SERPINB5-mediated GMPS degradation facilitated TP53 repression, which promoted the radioresistance of NPC cells. This novel working model related to TP53 suppression provided new insight into NPC radioresistence clinically. [ABSTRACT FROM AUTHOR]

The article presents a research which examines the effects of intermittent hypoxia (IH) on cerebellar granule cells and attempts to identify the mechanism of IH-induced cell death. According to the research, the process of IH increased oxidative stress and subsequently leading to cell death is partially mediated by poly (ADP-ribose) polymerase (PARP) activation .

Glycinergic synapses in photoreceptors are made by centrifugal feedback neurons in the network, but the function of the synapses is largely unknown. Here we report that glycinergic input enhances photoreceptor synapses in amphibian retinas. Using specific antibodies against a glycine transporter (GlyT2) and glycine receptor β subunit, we identified the morphology of glycinergic input in photoreceptor terminals. Electrophysiological recordings indicated that 10 μM glycine depolarized rods and activated voltage-gated Ca2+ channels in the neurons. The effects facilitated glutamate vesicle release in photoreceptors, meanwhile increased the spontaneous excitatory postsynaptic currents in Off-bipolar cells. Endogenous glycine feedback also enhanced glutamate transmission in photoreceptors. Additionally, inhibition of a Cl− uptake transporter NKCC1 with bumetanid effectively eliminated glycine-evoked a weak depolarization in rods, suggesting that NKCC1 maintains a high Cl− level in rods, which causes to depolarize in responding to glycine input. This study reveals a new function of glycine in retinal synaptic transmission. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) structural proteins (S, E, M, and NC) localize in different subcellular positions when expressed individually. However, SARS-CoV M protein is co-localized almost entirely with S, E, or NC protein when co-expressed in the cells. On the other hand, only partial co-localization was observed when S and E, S and NC, or E and NC were co-expressed in the cells. Interactions between SARS-CoV M and other structural proteins but not interactions between S and E, S and NC, or E and NC were further demonstrated by co-immunoprecipitation assay. These results indicate that SARS-CoV M protein, similar to the M proteins of other coronaviruses, plays a pivotal role in virus assembly. The cytoplasmic C-terminus domain of SARS-CoV M protein was responsible for binding to NC protein. Multiple regions of M protein interacted with E and S proteins. A model for the interactions between SARS-CoV M protein and other structural proteins is proposed. This study helps us better understand protein-protein interactions during viral assembly of SARS-CoV. [ABSTRACT FROM AUTHOR]

The article presents short notes on biomedical issues. In 1991 connective tissue growth factor (CTGF), a member of the CNN family of secreted, matrix-associated proteins, was discovered as a secreted protein in the conditioned media of cultured human umbilical vascular endothelial cells. Previous studies showed that T-box genes play a significant role in cell specification and amorphousness. In Taiwan, it has been found that chewing of betel quit causes oral cancer, which is the top sixth killer of all types of cancer.

Biomedical literature and database annotations, available in electronic forms, contain a vast amount of knowledge resulting from global research. Users, attempting to utilize the current state-of-the-art research results are frequently overwhelmed by the volume of such information, making it difficult and time-consuming to locate the relevant knowledge. Literature mining, data mining, and domain specific knowledge integration techniques can be effectively used to provide a user-centric view of the information in a real-world biological problem setting. Bioinformatics tools that are based on real-world problems can provide varying levels of information content, bridging the gap between biomedical and bioinformatics research. We have developed a user-centric bioinformatics research tool, called BioMap, that can provide a customized, adaptive view of the information and knowledge space. BioMap was validated by using inflammatory diseases as a problem domain to identify and elucidate the associations among cells and cellular components involved in multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE). The BioMap system was able to demonstrate the associations between cells directly excavated from biomedical literature for inflammation, EAE and MS. These association graphs followed the scale-free network behavior (average γ = 2.1) that are commonly found in biological networks. [ABSTRACT FROM AUTHOR]

Some intermediate filament (IF) proteins expressed in the development of glia include nestin, vimentin, and glial fibrillary acidic protein (GFAP). However, GFAP is the major intermediate filament protein of mature astrocytes. To determine the organization of GFAP in glial cells, rat GFAP cDNA tagged with enhanced green fluorescent protein (EGFP) was transfected into the rat C6 glioma cell line. After selection, two stable C6-EGFP-GFAP cell lines were established. Stable C6-EGFP-GFAP cell lines with or without heat shock treatment were analyzed by immunocytochemistry, electron microscopy, and Western blot analysis. In the transient transfection study, EGFP-GFAP transiently expressed in C6 cells formed punctate aggregations in the cytoplasm right after transfection, but gradually a filamentous structure of EGFP-GFAP was observed. The protein level of nestin in the C6-EGFP-GFAP stable clone was similar to that in the pEGFP-C1 transfected C6 stable clones and non-transfected C6 cells, whereas the level of vimentin was reduced in Western blotting. Interestingly, the expression level of small heat shock protein αB-crystallin in C6-EGFP-GFAP cells was also enhanced after transfection. Immunostaining patterns of C6-EGFP-GFAP cells showed that GFAP was dispersed as a fine filamentous structure. However, after heat shock treatment, GFAP formed IF bundles in C6-EGFP-GFAP cells. In the meantime, αB-crystallin also colocalized with IF bundles of GFAP in C6-EGFP-GFAP cells. The heat-induced GFAP reorganization we found suggested that small heat shock protein αB-crystallin may play a functional role regulating the cytoarchitecture of GFAP. [ABSTRACT FROM AUTHOR]

Inhibitory effects of ethanolic extracts from 10 Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were investigated. By a bioassay-guided fractionation procedure, NN-B-5 was identified from seeds of N. nucifera. NN-B-5 significantly blocked HSV-1 multiplication in HeLa cells without apparent cytotoxicity. To elucidate the point in HSV-1 replication where arrest occurred, a set of key regulatory events leading to the viral multiplication was examined, including HSV-1 DNA synthesis and viral immediate early gene expressions. Data from polymerase chain reaction and Southern blotting showed that there were impairments of HSV-1 DNA replication in HeLa cells treated with NN-B-5. Results indicated that the production and mRNA transcription of infected cell protein (ICP) 0 and ICP4 were decreased in NN-B-5 treated HeLa cells. Results of an electrophoretic mobility shift assay demonstrated that NN-B-5 interrupted the formation of α- trans-induction factor/C1/Oct-1/GARAT multiprotein/DNA complexes. The mechanisms of antiviral action of NN-B-5 seem to be mediated, at least in part, through inhibition of immediate early transcripts, such as ICP0 and ICP4 mRNA and then blocking of all downstream viral products accumulation and progeny HSV-1 production. [ABSTRACT FROM AUTHOR]

The P2X7 nucleotide receptor is an ATP-gated ion channel that plays an important role in bone cell function. Here, we investigated the effects of L-tyrosine derivatives 1–3 as potent P2X7 antagonists on human primary osteoclasts. We found that the level of expression of P2X7 receptor increased after treatment with the derivatives 1–3, together with the induction of high levels of apoptosis. This effect is associated with activation of caspase-3 and inhibition of expression of IL-6. Interestingly, no pro-apoptotic effect of compounds 1–3 was found on human osteoblasts. Our results suggest that the development of specific P2X7 receptor antagonists may be considered a useful tool to modulate apoptosis of human osteoclasts. Since bone loss due to osteoclast-mediated resorption represents one of the major unsolved problem in osteopenic disorders, the identification of molecules able to induce apoptosis of osteoclasts is of great interest for the development of novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Reactive oxygen species produced by NADPH oxidase appear to play a role in the response of human lung fibroblast cells to rhinovirus infection. The purpose of the following studies was to characterize the NADPH oxidase components in these cells, to examine the effect of rhinovirus challenge on the expression of these proteins, and to confirm previous studies suggesting a role for p47-phox in the oxidant response to rhinovirus challenge. The results revealed that the NADPH oxidase components p47-phox, p67-phox, p22-phox, and NOX4 were expressed in lung fibroblast cells. In contrast, gp91-phox was not expressed in this cell line. Expression of p67-phox was upregulated by rhinovirus challenge. The functional role of NADPH oxidase in the rhinovirus-induced oxidant stress and elaboration of IL-8 was confirmed by detection of significant reductions in oxidant stress and IL-8 elaboration following transfection of the cells with antisense nucleotides to p47-phox. The lack of gp91- phox in cultured lung fibroblast cells, the induction of p67-phox by rhinovirus, and the confirmation of participation of p47-phox in rhinovirus-induced oxidant stress are significant findings of this study and form a basis for future investigations into understanding the mechanisms of the NADPH oxidase response to rhinovirus infection. Copyright © 2004 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In this study, we examined the postnatal expression patterns of p63 and other keratinocyte stem cell markers in the rat cornea in an attempt to determine the markers that best represent characteristics of corneal keratinocyte stem cells. We show that the expression of p63 in the rat cornea is unique and differs from that observed in humans. It changes with age, from central cornea-positive, peripheral cornea-positive, and limbus-positive, to central cornea-positive, peripheral cornea-positive, and limbus-negative, and finally to central cornea-negative, peripheral cornea-positive, and limbus-negative, as examined by immunohistochemical staining. However, when a more sensitive staining method was used, the limbus was also shown to be positive for p63, indicating a lower level of expression than that of the peripheral cornea. The basal layer of the rat limbal epithelium is the site where β-catenin+, K14+, PCNA–, and K3– cells reside. This cell layer is also the site where slow-cycling cells are located. In contrast with observations made in humans, our results clearly indicate that p63 is expressed in stem cells and young transient amplifying cells of the rat cornea, with higher levels of expression in the latter. Copyright © 2004 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Neuronal transplantation has provided a promising approach for treating neurodegenerative diseases. Recently, efforts have been directed at in vitro induction of various stem cells to transform into neurons. We report the first successful quantities in an in vitro attempt at directing the transformation into neurons of human umbilical mesenchymal cells, which are capable of rapid proliferation in vitro and are easily available. When cultured in neuronal conditioned medium, human umbilical mesenchymal cells started to express neuron-specific proteins such as NeuN and neurofilament (NF) on the 3rd day and exhibited retraction of the cell body, elaboration of processes, clustering of cells and expression of functional mRNA responsible for the synthesis of subunits of the kainate receptor and glutamate decarboxylase on the 6th day. Between the 9th and 12th days, the percentage of human umbilical mesenchymal cells expressing NF was as high as 87%, while functionality was demonstrated by glutamate invoking an inward current. At this stage, cells were differentiated into mature neurons in the postmitosis phase. Copyright © 2004 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We have used the human calcium- and temperature-dependent (HaCaT) keratinocyte cell line to elucidate mechanisms of switching from a proliferating to a differentiating state. When grown in low calcium medium (<0.1 mM) HaCaT cells proliferate. However, an increase in the calcium concentration of the culture medium, [Ca2+]0, induces growth arrest and the cells start to differentiate. Numerous studies have already shown that the increase in [Ca2+]0 results in acute and sustained increases in intracellular calcium concentration, [Ca2+]i. We find that the Ca2+-induced cell differentiation of HaCaT cells is also accompanied by a significant decrease in mitochondrial membrane potential, DeltaPsi. By combining patch-clamp electrophysiological recordings and microspectrofluorimetric measurements of DeltaPsi on single cells, we show that the increase in [Ca2+]i led to DeltaPsi depolarization. In addition, we report that tetraethylammonium (TEA), a blocker of plasma membrane K+ channels, which is known to inhibit cell proliferation, and 4,4’-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), a blocker of plasma membrane Cl– channels, also affect DeltaPsi. Both these agents stimulate HaCaT cell differentiation. These data therefore strongly suggest a direct causal relationship between depolarization of DeltaPsi and the inhibition of proliferation and induction of differentiation in HaCaT keratinocytes. Copyright © 2004 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Infection of bovine arterial endothelial (BAE) cells with bovine herpesvirus-4 (BHV-4) induced biphasic activation of one of the cellular mitogen-activated protein kinase (MAPK) downstream targets, extracellular signal-regulated kinase (ERK). ERK activity reached a maximum within 0.5 h postinfection (h.p.i.), and had declined and returned to basal levels by 2 h.p.i. However, at 18– 24 h.p.i., a second phase of increased ERK activation occurred. Treatment of BHV-4-infected BAE cells with either U0126, a potent inhibitor of MAPK/ERK kinase, or arsenite dose-dependently blocked ERK activation and inhibited viral DNA synthesis and viral replication in the culture. Further detailed investigations revealed that transcription of viral immediate-early gene 2 (IE-2), which is required for viral DNA replication, was significantly suppressed by both U0126 and arsenite. These results imply that ERK activation may play a pivotal role in herpesvirus replication, and that inhibition of ERK activation can effectively inhibit viral IE protein synthesis and viral replication. Copyright © 2004 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Hyperglycemia and mannitol activate protein kinase C (PKC) and induce mesangial cell hypocontractility that subsequently may modulate renal function. Since focal adhesion kinase (FAK) activation is known to be linked with PKC activity, FAK may also be involved in mesangial cell contraction. To facilitate our understanding of the PKC- and FAK-modulating mechanism, we developed an in vitro model of mouse mesangial cell hypocontractility induced by hyperglycemia or mannitol. Mouse mesangial cells (CRL-1927) were exposed to: normal D -glucose (group N), high D -glucose (group H), and control groups at the same osmolality as H plus L -glucose (group L) and mannitol (group M). Changes in the planar surface area of cells in response to 1 μM phorbol 12-myristate 13-acetate (PMA) were determined. Western blot analyses for PKC, phosphorylated (p)-PKC, tyrosine phosphorylation, FAK, and p-FAK were done on each of these four groups. The effects of mannitol in various doses on cell contraction and activation of PKC and FAK were also assayed. The planar surface areas of groups H and M both showed an attenuated change in response to PMA stimulation. Before PMA stimulation, the baseline PKC expression of groups H and M showed a higher expression of p-PKCα and δ than that seen in group N (p < 0.05). Results of tyrosine phosphorylation and immunoprecipitation showed that FAK may be involved in this contraction process. The total amount of FAK showed no significant difference among the four experimental groups; however, p-FAK was found to have significantly increased in group M (p < 0.05). The use of PKC and tyrosine kinase inhibitors reduced PMA-induced mesangial cell contraction in all four groups. Activation of PKCα, δ, and FAK with the resultant inhibition of mesangial cell contraction by mannitol was found to be dose-dependent. These results may provide a correlation between increased expression of several PKC isoforms and, in particular, increased phosphorylation levels of PKCα and δ and hypocontractility induced by high glucose and mannitol treatment. Furthermore, the mannitol-induced hypocontractility involving PKC and FAK occurred in a dose-dependent manner. Copyright © 2004 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Current criteria for the diagnosis of malignant GISTs do not always reliably predict patient outcomes. In order to search for genetic markers with prognostic potential, chromosomal imbalance aberrations (CIAs) were analyzed in 28 subjects with GIST using comparative genomic hybridization and correlated with clinicopathological features. Except for a small rectal tumor, CIAs were identified in all GISTs, including 14 from the stomach, 11 from the small intestine, 1 from the esophagus, and 1 from the rectum. Losses were more common than gains. The median number of CIAs in high-risk GISTs was significantly higher than that in low-risk GISTs (5.60 ± 2.59 vs. 3.38 ± 2.55; p < 0.05), especially for losses (4.60 ± 1.84 vs. 2.63 ± 2.13; p < 0.01). Loss of 14q was the most common CIA in both low-risk and high-risk GISTs, and can be regarded as an early event of GIST development. Losses of 1p and 15q were also very common, often coexisting, and were slightly more frequent in high-risk GISTs than in low-risk GISTs. Other recurrent CIAs, including losses of 10q, 13q, 15q, 18q, and 22q and gains of 5p, 12q, 17q, and 20q were relatively less common in this series. Among these CIAs, losses of 13q, 10q (with minimal overlapping on q11–q22), and 22q were most likely the chromosomal loci potentially harboring the tumor suppressor gene(s) which may be related to early recurrence and/or metastasis during malignant transformation of GISTs. Copyright © 2004 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Two sulfonamide derivatives of porphycene, namely PS6 and PS6A, were synthesized, and their photodynamic efficacies on the nasopharyngeal carcinoma (NPC) cell line NPC/CNE-2 were evaluated. By comparing the 50% lethal concentrations (LC[sub50]) of these photosensitizers, we found that PS6A with a cationic ammonium group on the side chain exhibited potent photocytotoxicity on the NPC cell line. At a light dose of 1 J/cm² the LC[sub50] values of PS6 and PS6A for NPC cells were 11.6 and 1.92 µM, respectively. CNE-2 was found to rapidly take up PS6A in the first hour of incubation, and the uptake kinetics steadily increased to a plateau level after 18 h of incubation. The uptake of PS6A was temperature dependent. Over 99% of CNE-2 cells were sensitized by PS6A 24 h after drug treatment. Collapse of the mitochondrial membrane potential was also observed in PS6A photodynamic therapy (PDT)-treated CNE-2 cells 1.5 h after PDT. Confocal microscopy revealed that PS6A was predominantly localized in the mitochondria, lysosomes and Golgi bodies of NPC cells. Significant genotoxicity was not observed in CNE-2 cells. In functional studies, the in vitro formation of a capillary-like network of human umbilical vein endothelial cells in Matrigel was greatly inhibited by PS6A PDT in a dose-dependent manner. In conclusion, PS6A mediates both in vitro antitumor and antiangiogenic activities. PS6A might be a candidate for photodynamic treatment of NPCs. [ABSTRACT FROM AUTHOR]

Whether reactive oxygen species (ROS) mediate β-amyloid (Aβ) neurotoxicity remains controversial. Naive PC12 cells (PC12) and nerve growth factor-differentiated PC12 cells (dPC12) were used to study the role of ROS in cell death induced by Aβ[sub25-35]. The viability of PC12 and dPC12 cells decreased by 30-40% after a 48-hour exposure to 20 μM Aβ[sub25-35]. Microscopic examination showed that Aβ[sub25-35] induced necrosis in PC12 cells and apoptosis in dPC12 cells. Vitamin E (100 μM) and other antioxidants protected PC12 cells, but not dPC12 cells, against the cytotoxic effect of Aβ[sub25-35]. Since H[sub2]O[sub2] has been proposed to be involved in Aβ toxicity, the effects of H[sub2]O[sub2] on PC12 and dPC12 cells were studied. Differentiated PC12 cells appeared to be significantly more resistant to H[sub2]O[sub2] than naive PC12 cells. These data suggest that ROS may mediate Aβ[sub25-35} toxicity in PC12 cells but not in dPC12 cells. Because the intracellular levels of ROS were elevated during the differentiation of PC12 cells, the baseline levels of ROS in these two model cell types may determine the intracellular mediators for Aβ[sub25-35] toxicity. Therefore, the protective effects of antioxidants against Aβ may depend upon the redox state of the cells. [ABSTRACT FROM AUTHOR]

The aims of the present study were twofold: (1) simultaneous determinations of Na[sup+] transport parameters of erythrocytes from 40 healthy donors and 28 septic patients as assessed by a score of severity of sepsis (SSS), and (2) examination of the correlation between the SSS and specific Na[sup+] transport abnormalities. Erythrocytes were obtained and loaded with different ionic compositions and cellular Na[sup+] contents before determination of the near-maximal Na[sup+] pump rate (Vmax), the physiological extrusion rate of Na[sup+] (v) and the number of ouabain-binding sites (Bmax). In erythrocytes from septic patients, the cellular Na[sup+] content was 28% higher (p < 0.001), with no differences in water content compared to erythrocytes from healthy donors. This elevated Na[sup+] content was accompanied by significantly higher values for Vmax (43%), v (24%) and Bmax (48%) of the Na[sup+] pump in septic erythrocytes. Moreover, significant positive correlations existed between Vmax and SSS (p = 0.028) and between cellular Na[sup+] content and SSS (p = 0.005). These data suggest that during sepsis, membrane alterations occur and result in an increased cellular Na[sup+] content. Active Na[sup+] transport (Vmax and v) was significantly stimulated, possibly as a consequence of a secondary response to the elevated Na[sup+] of cells. Both cellular Na[sup+] and Vmax correlated well with the severity of sepsis, suggesting that these altered transport parameters may reflect the progress of sepsis. [ABSTRACT FROM AUTHOR]

Excessive proliferation of vascular smooth muscle cells (VSMC) in the intima is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. Therefore, control of VSMC growth may be a suitable therapeutic intervention in vascular proliferative disorders. In the present work, we have studied the 2-benzyloxybenzaldehyde (CCY1a)-mediated antiproliferative effect and its mechanisms of action. CCY1a inhibited serum-induced VSMC proliferation in a concentration-dependent manner, as demonstrated using [[sup 3] H]thymidine incorporation and MTT assays; the IC[sub 50] values were calculated to be 7.0 × 10[sup –6] and 1.2 × 10[sup –5] M, respectively. Furthermore, it also significantly suppressed serum-induced progression of the cell cycle, as shown by flow cytometric analysis. CCY1a as well as PD98059 almost completely abolished serum-induced activation of p42/44 mitogen-activated protein kinase (MAPK), the downstream effectors of c-fos and c-jun mRNA expression and activator protein-1 (AP-1) DNA binding activity, suggesting the central roles of these signaling cascades. Interestingly, CCY1a also effectively blocked serum-induced IκB-α phosphorylation, IκB-α degradation and nuclear factor-κB (NF-κB) binding activity. Based on these observations, we examined the effect of CCY1a on serum-mediated Ras activity, an upstream regulator of the above signaling events. The data demonstrated a marked inhibition of Ras activation by CCY1a. We conclude that CCY1a blocks cell proliferation via inhibition of the upstream effector of Ras and downstream events, including p42/44 MAPK activation and c-fos and c-jun mRNA expression, as well as NF-κB and AP-1 DNA binding activities.Copyright © 2002 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We have recently demonstrated that alteration of the human immunodeficiency virus type 1 (HIV-1) Gag/GagPol ratio in virus-producing cells reduces the infectivity of progeny viruses and hinders the formation of stable virion RNA dimers without impairing virion packaging of the viral genomic RNA. In addition, we have previously shown that the expression of GagPol mediates the selective packaging of tRNA[sup Lys3] . In this study we report that overexpression of uncleaved GagPol in the virus-producing cell did not alter the packaging levels of tRNA[sup Lys3] . Similarly, altering the virion-associated Gag/GagPol ratio did not affect the virion packaging of the HIV-1 envelope protein nor cyclophilin A. Thin section electron microscopy analysis of the cells overexpressing protease-defective [PR(–)] GagPol revealed immature virions but no mature virions. These immature virions were seen both extracellularly and in membrane-bound cytoplasmic vacuoles. Furthermore, an accumulation of electron-dense material was occasionally found at the plasma membrane and associated with intracytoplasmic membranous vacuoles in cells expressing excess PR(–) GagPol. No intracellular HIV was seen in the wild-type control. Density gradient analysis showed that the overall density of these mutant virions with excess PR(–) GagPol was identical to that of the wild-type HIV-1. The findings indicate that overexpression of PR(–) GagPol, in the presence of Gag synthesis, promotes intracellular budding of the mutant virions and inhibits virus maturation.Copyright © 2002 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenium-dependent glutathione peroxidase, can interact with lipophilic substrates, including phospholipid hydroperoxides, fatty acid hydroperoxides and cholesterol hydroperoxides, and can reduce them to hydroxide compounds. It also seems to be a major regulator of lipid oxygenation in human epidermoid carcinoma A431 cells. In order to study the functional role of PHGPx in the regulation of 12-lipoxygenase and cyclooxygenase, cDNA of PHGPx was inserted into pcDNA3.1/His, and a plasmid designated as S4 with the His-tag sequence inserted between PHGPx and its 3′-untranslated region was constructed. A number of stable transfectants of A431 cells that could express the tag-PHGPx were generated using plasmid S4. Using an intact cell assay system, the metabolism of arachidonic acid to prostaglandin E[sub 2] significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line. If the intact cell assay was carried out in the presence of 13-hydroperoxyoctadecadienoic acid as a stimulator of lipid peroxidation, formation of 12-hydroxyeicosatetraenoic acid from arachidonic acid also significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line, indicating that PHGPx could downregulate the 12-lipoxygenase activity in cells. These results support the hypothesis that PHGPx plays a pivotal role in the regulation of arachidonate metabolism in A431 cells.Copyright © 2002 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Recognition and incision of UV-DNA adducts play key roles in the efficacy of nucleotide excision repair. Damaged-DNA recognition activity has been identified from primate cells as a complex of DDB1 (127-kD) and DDB2 (48-kD) subunits. However, the function of damaged-DNA binding proteins (DDBs) in damaged-DNA recognition is not well understood. To assess the functional correlation between DDBs and UV-damaged-DNA recognition activity, we identified UV-damaged-DNA recognition activities in rodent cell lines. There is a cell type-dependent expression of DDB1 and DDB2. Rodent cells had less abundant DDBs and lower UV-damaged-DNA recognition activity than did human tumor cells. Interestingly, the profusion of DDBs is associated with UV-damaged-DNA recognition activity in these cell lines. We also discovered tissue-dependent expression of DDBs and its functional correlation with UV-damaged-DNA recognition activity. cDNA (3850 nucleotides) from rat ddb1 was isolated. It contained the complete length of the open reading frame that encodes an 1140-amino-acid polypeptide with a predicted molecular weight of 126.8 kD. The predicted protein size from the rat ddb1 gene resembles that from human DDB1 (127 kD). Rat DDB1 shares highly conserved sequencing (greater than 98% similarity) with those of mouse, human, and monkey. Rat and fruit fly DDB1 exhibit 62.23% identity and 57.66% homology. The evolutionary conservation of the DDB1 sequence suggests that DDB1 may play a pivotal role in mammals as well as in other eukaryotes. However, overexpression of DDB1 did not augment UV-damaged-DNA recognition activity in human HeLa, hamster V79, or rat PC12 cells. In contrast, restricting DDB2 expression by antisense ddb2 partially inhibited UV-damaged-DNA recognition activity in cells, whereas overexpressing DDB2 through a recombinant ddb2 adenovirus partly restored the recognition activity of these cells. These findings support the notion that DDB abundance is functionally correlated with UV-damaged-DNA recognition activity. These results also suggest that the profusion of DDB2, but not DDB1, may moderate UV-damaged-DNA recognition activity.Copyright © 2002 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The ability of cells of the human monocyte/macrophage lineage to host HIV-1 replication while resisting cell death is believed to significantly contribute to their ability to serve as a reservoir for viral replication in the host. Although macrophages are generally resistant to apoptosis, interruption of anti-apoptotic pathways can render them susceptible to apoptosis. Here we report that HIV-1[sub BAL ] infection of primary human monocyte-derived macrophages (MDM) upregulates the mRNA and protein levels of the anti-apoptic gene, Bcl-2. Furthermore, this upregulation can be quantitatively mimicked by treating MDM with soluble HIV-1 Tat-86 protein. These results suggest that in infecting cells of the monocyte/macrophage lineage, HIV-1 may be benefiting from additional protection against apoptosis caused by specific upregulation of cellular anti-apoptotic genes.Copyright © 2002 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Chronic and excessive ethanol consumption is known to alter neuron and glial cell functions in the central nervous system (CNS). Astrocytes comprise the major cell type in the brain. These immune active cells are capable of responding to proinflammatory cytokines and endotoxins, which stimulate transcriptional pathways leading to induction of genes, including the inducible nitric oxide synthase (iNOS) and secretory phospholipase A[sub 2 ] (sPLA[sub 2] ). In this study, we investigate the effects of ethanol on cytokine-induced iNOS and sPLA[sub 2] in immortalized astrocytes (DITNC). When DITNC cells were exposed to ethanol (0–200 mM) for 4 h prior to subsequent stimulation with cytokines for 16 h, NO production decreased with increasing ethanol concentrations starting from 50 mM. At ethanol concentrations higher than 100 mM, ethanol also inhibited cytokine-induced sPLA[sub 2] release into the culture medium. The inhibitory effect of ethanol on NO production corresponds well with the decrease in iNOS protein and NOS enzyme activity, but not with iNOS and sPLA[sub 2] mRNA nor binding of NF-κB to DNA. The inhibition of cytokine-induced NO production by ethanol was also dependent on the time of ethanol exposure to the cells, but addition of acetaldehyde up to 200 μM did not elicit any changes. Taken together, these results provide evidence for a posttranscriptional mode of ethanol action on the cytokine induction pathway for NO production in astrocytes.Copyright © 2001 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Objective: The purpose of this study was to evaluate the contribution of capacitative calcium influx to intracellular calcium levels during agonist-induced stimulation of vascular smooth muscle cells. Methods: Aortic vascular smooth muscle cells (A7r5) were loaded with Indo-1 and intracellular calcium transients were measured. Cells were challenged with either arginine vasopressin (0.5 μM) or thapsigargin (1 μM). Lanthanum (1 mM) was used to block capacitative calcium influx through store-operated channels. Calcium traces were analyzed for basal, peak and plateau responses. Recordings were derivatized and integrated to gain additional information. Nonlinear regression provided a time constant that describes restoration of ionic equilibrium involving both sequestration and extrusion pathways. Results: Stimulation of cells with thapsigargin produced a non-L-type calcium influx that was attenuated by lanthanum. Cells excited with vasopressin exhibited a rapid calcium increase followed by a gradual decrease to a plateau level. Lanthanum pretreatment prior to stimulation caused no significant change in baseline, peak or plateau calcium levels as compared to control. Lanthanum caused no significant change in maximal calcium release rate, calcium integrals or time constant as compared to control. Conclusions: Capacitative calcium entry can occur in vascular smooth muscle cells, but does not appear to contribute significantly to the vasopressin response.Copyright © 2000 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Thapsigargin (TG), an endoplasmic reticular (ER) Ca[sup 2+] -ATPase inhibitor, can increase the intracellular calcium concentration and then deplete the TG-sensitive intracellular Ca[sup 2+] pool. In this study, we investigated the effects of TG on cell viability and tumor necrosis factor-α (TNF-α) production in the murine macrophage RAW 264.7 cell line. We found that treatment with TG (10–800 nM) induced apoptosis in RAW 264.7 cells in a dose-dependent manner (IC[sub 50] , 200 nM). Lipopolysaccharide (LPS, 1 μg/ml) markedly potentiated low concentrations of TG (10–75 nM) in inducing apoptosis (IC[sub 50] , 20 nM) as revealed by the DNA ladder. Polymycin B (an LPS receptor antagonist) inhibited the cytotoxic effect induced by LPS plus TG. Although TG, A23187 and ionomycin all definitely increased intracellular Ca[sup 2+] concentrations, neither A23187 nor ionomycin mimicked TG in inducing apoptotic events in LPS-activated RAW 264.7 cells. Moreover, the production of TNF-α induced by LPS was profoundly potentiated by TG but not by A23187 or by ionomycin. We conclude from these combined results that TG-sensitive ER Ca[sup 2+] stores play a pivotal role in modulating cell viability and TNF-α production. The mutual potentiation between the LPS receptor signaling pathway and the depletion of ER Ca[sup 2+] stores implies the existence of cross-talk between these multiregulatory mechanisms in this murine macrophage RAW 264.7 cell line.Copyright © 2000 National Science Council, ROC and S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

(+)-MK801, a noncompetitive NMDA receptor antagonist, was reported to exhibit anticonvulsive and neuroprotective activities during the postischemic period. Intravenous administration of (+)-MK801 produced tachycardia in rats, but bradycardia in pigs. We examined the mechanical and electrophysiological effects of (+)-MK801 on rat cardiac tissues. (+)-MK801 dose-dependently increased (3–100 μM) twitch tension in rat atria and ventricular strips. The spontaneous beating rate in rat right atria, however, was dose-dependently decreased by (+)-MK801. The inotropic effect of (+)-MK801 was affected neither by α[sub 1] -antagonist (1 μM prazosin) nor by β[sub 1] -adrenoceptor antagonist (3 μM atenolol), but significantly by a transient outward K[sup +] channel blocker (3 mM 4-aminopyridine). (+)-MK801 did not cause any significant change of intracellular cAMP content. Electrophysiological study in rat ventricular cells revealed that (+)-MK801 concentration-dependently prolonged the action potential duration with a concomitant decrease in the maximum rate of the action potential upstroke (V[sub max] ) and an increase in the recovery time constant of V[sub max] . Voltage clamp study showed that (+)-MK801 (3 μM) reduced inward Na[sup +] current (I[sub Na] ), along with a slowing of its recovery from inactivation and a slight negative shift of its voltage-dependent steady-state inactivation curves. At a much higher concentration (30 μM), (+)-MK801 slightly reduced the amplitude of L-type calcium inward current (I[sub Ca] ), although the voltage dependence of its steady-state inactivation was unaffected. For the potassium currents in rat ventricular cells, 3 μM of (+)-MK801 reduced the peak transient outward current (I[sub to] ), steady-state outward current (I[sub ss] ) and inward current through K[sub 1] channels. The inhibition of I[sub to] was associated with a prominent negative shift in the voltage dependence of its steady-state inactivation curve. The outward current through K[sub 1] channels was unaffected. These results indicate that (+)-MK801 may be a strong I[sub Na] and I[sub to] blocker with some I[sub Ca] blocking activity. The inhibition of I[sub to] and other K[sup +] efflux would prolong action potential duration, produce positive inotropic action and contribute to the negative chronotropic effect of (+)-MK801. [ABSTRACT FROM AUTHOR]

The mechanisms by which different concentrations of cesium modify membrane potentials and currents were investigated in guinea pig single ventricular myocytes. In a dose-dependent manner, cesium reversibly decreases the resting potential and action potential amplitude and duration, and induces a diastolic decaying voltage tail (V[sub ex] ), which increases at more negative and reverses at less negative potentials. In voltage-clamped myocytes, Cs[sup +] increases the holding current, increases the outward current at plateau levels while decreasing it at potentials closer to resting potential, induces an inward tail current (I[sub ex] ) on return to resting potential and causes a negative shift of the threshold for the inward current. During depolarizing ramps, Cs[sup +] decreases the outward current negative to inward rectification range, whereas it increases the current past that range. During repolarizing ramps, Cs[sup +] shifts the threshold for removal of inward rectification negative slope to less negative values. Cs[sup +] -induced voltage and current tails are increased by repetitive activity, caffeine (5 mM) and high [Ca[sup 2+] ][sub O] (8.1 mM), and are reduced by low Ca[sup 2+] (0.45 mM), Cd[sup 2+] (0.2 mM) and Ni[sup 2+] (2 mM). Ni[sup 2+] also abolishes the tail current that follows steps more positive than E[sub Ca] . We conclude that Cs[sup +] (1) decreases the resting potential by decreasing the outward current at more negative potentials, (2) shortens the action potential by increasing the outward current at potentials positive to the negative slope of inward rectification, and (3) induces diastolic tails through a Ca[sup 2+] -dependent mechanism, which apparently is an enhanced electrogenic Na-Ca exchange. [ABSTRACT FROM AUTHOR]

In this study, the effects of carbachol (CCh) on twitch tension, intracellular Na[sup +] activity (a[sup i] [sub Na] ), and action potential were simultaneously measured in canine cardiac Purkinje fibers in order to examine the regulation of inotropy through muscarinic receptors and its relation to a[sup i] [sub Na] . In fibers driven at 1 Hz, CCh (10 μM) initially and transiently decreased and then increased the twitch tension by 36 ± 8%. The action potential showed a significant elevation of the plateau and a significant shortening of the duration at 90% repolarization (APD[sub 90] ), from 403 ± 7 to 389 ± 7 ms. The a[sup i] [sub Na] decreased from 7.4 ± 0.4 to 6.7 ± 0.3 mM (n = 23, p < 0.05). Atropine (1 μM) decreased the twitch tension by 21 ± 6% (n = 7, p < 0.05) without significant effects on the action potential and a[sup i] [sub Na] , and inhibited the effects of CCh. Cs[sup +] (20 mM) increased the plateau height and APD[sub 90] , enhanced the twitch tension by 66 ± 24%, but decreased a[sup i] [sub Na] from 7.3 ± 0.3 to 6.3 ± 0.4 mM (n = 6, p < 0.05). In the presence of 20 mM Cs[sup +] , some fibers generated slow responses. The addition of 10 μM CCh further increased the twitch tension and APD[sub 90] , and decreased a[sup i] [sub Na] from 6.3 ± 0.4 to 5.3 ± 0.3 mM. Ouabain (0.3 μM) increased the twitch tension and a[sup i] [sub Na] , and inhibited the CCh-induced decrease of a[sup i] [sub Na] . In the presence of ouabain, 20 mM Cs[sup +] depolarized the fiber and generated slow responses with a decreased a[sup i] [sub Na] . The addition of 10 μM CCh enhanced the slow action potential, and increased a[sup i] [sub Na] although there was a transient decrease during early exposure. These results suggest that activation of muscarinic receptors in canine Purkinje fibers results in an enhancement of the Na[sup +] -K[sup +] pump activity and a biphasic inotropic response, probably via different receptor subtypes. The inhibitory effect, most likely through M[sub 2] receptors, is associated with the activation of K[sup +] channels. The stimulatory effect, on the other hand, is probably due to the action on the M[sub 1] receptors, resulting in increases in Ca[sup 2+] currents. [ABSTRACT FROM AUTHOR]

T cells with CD4–CD8– (double negative, DN) phenotype in MRL-lpr/lpr mouse serve as a model to establish the correlation between the extremely low IL-2 gene expression and the specific signaling inactivation. The extent of nonresponsiveness in lpr DN cells was distinctive in several unusual defects. First, the poor IL-2 production in lpr DN cells could not be restored by supplement of signals known to augment IL-2 response in normal T cells. Second, the activations of both mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK) were attenuated in lpr DN cells upon direct activation by TPA/A23187. Third, IL-2 mRNA was degraded much faster in lpr DN cells than that in normal T cells. Fourth, of the four major transcriptional elements on IL-2 promoter, only AP-1 and nuclear factor of activated T cells (NFAT)-binding activities were suppressed in lpr DN T cells. Altogether, these results suggest that an extremely low level of IL-2 production in lpr DN T cells was due to both the increased instability of mRNA and the reduced activation of IL-2 gene promoter, the latter defect could be attributed to the inactivation of AP-1 and NF-AT as well as the poor activation of the upstream MAP kinase and JNK. [ABSTRACT FROM AUTHOR]