The effects of glaucoma on the latency of the multifocal visual evoked potential.

AIMS: To determine the effect of glaucomatous damage on the latency of the multifocal visual evoked potential (mfVEP). METHODS: Monocular mfVEPs were recorded from a glaucoma group (n = 50) defined by a glaucomatous disc and an abnormal visual field and a control group (n = 47). 25 patients were characterised as normal tension glaucoma (NTG) and 25 as high tension glaucoma (HTG). Monocular and interocular latency analyses of the more affected eye were obtained using custom software. RESULTS: On interocular analysis, both the HTG and NTG groups showed a statistically significant increase in mean mfVEP latency with average relative latencies and percentage of points with significant delays of 1.7 ms and 10.3% (HTG) and 1.3 ms and 8.2% (NTG) compared to -0.3 ms and 2.7% (controls). On monocular analysis, only the HTG group showed a significant increase in latency with measures of 5.7 ms and 14.6% (HTG) compared to 3.2 ms and 10.6% (NTG) and 2.1 ms and 9.6% (controls). Using the 95th percentile of a normative group as the cut off, the sensitivity ranged from 20% to 38% and the specificity from 87% to 100% with the interocular analysis providing the best discrimination, CONCLUSION: Although up to 40% of patients showed delays in the mfVEP latency, these delays were modest, on average a few milliseconds. These results differ markedly from those of a recent conventional VEP study, which reported 100% sensitivity, 100% specificity, and an average delay that exceeded 25 ms.

The tree shrews: adjuncts and alternatives to primates as models for biomedical research.

The tree shrews are non-rodent, primate-like, small animals. There is increasing interest in using them to establish animal models for medical and biological research. This review focuses on the use of the tree shrews in in vivo studies on viral hepatitis, hepatocellular carcinoma (HCC), myopia, and psychosocial stress. Because of the susceptibility of the tree shrews (Tupaia belangeri) and their hepatocytes to infection with human hepatitis B virus (HBV) in vivo and in vitro, these animals have been used to establish human hepatitis virus-induced hepatitis and human HBV- and aflatoxin B1-associated HCC models. As these animals are phylogenetically close to primates in evolution and have a well-developed visual system and color vision in some species, they have been utilized to establish myopia models. Because dramatic behavioral, physiological, and neuroendocrine changes in subordinate male tree shrews are similar to those observed in depressed human patients, the tree shrews have been successfully employed to experimentally study psychosocial stress. However, the tree shrews holds significant promise as research models and great use could be made of these animals in biomedical research.

Microsatellite alterations in human hepatocellular carcinoma infected with hepatitis B virus: associated with the elevation of serum alpha-fetoprotein.

Identification of the basic genetic changes in human hepatocellular carcinoma (HCC) is very important for the understanding of this cancer. In this study, genomic DNA from 29 pairs of HCC and corresponding non-tumour tissues infected with hepatitis B virus (HBV) was prepared. Five CA-repeated microsatellite markers, including D8S277, D3S1029, D5S409, D2S123, and TP53, were used to analyse microsatellite alterations and their subtypes in these patients by polymerase chain reaction (PCR) amplification and denatured polyacrylamide gel electrophoresis. Microsatellite alterations were found in 15 of the 29 HCC patients (51.72%), implying that microsatellites are unstable in genomic DNA of HBV-infected HCC. It was found that frequency of microsatellite alterations in these HCC patients was not associated with patients' age, sex, status of tumour differentiation, and tumour size. Frequency of microsatellite alterations in HCC patients with cirrhosis tended to be less than that in patients without cirrhosis, but Fisher's exact test, 2-tailed, showed that this difference was not significant. Significantly more microsatellite alterations in serum alpha-fetoprotein (AFP)-positive cases were observed than those in serum AFP-negative ones, implying that the elevation of AFP in HBV-infected HCC may be associated with microsatellite stability.

Modulation of cisplatin cytotoxicity and cisplatin-induced DNA cross-links in HepG2 cells by regulation of glutathione-related mechanisms.

Glutathione (GSH), glutathione S-transferase (GST), and glutathione conjugate export pump (GS-X pump) have been shown to participate collectively in the detoxification of many anticancer drugs, including cisplatin. Identification and regulation of the rate-limiting step in the overall system for cisplatin detoxification is of crucial importance for sensitization of human tumor cells to cisplatin. In this study, the GSH content, GST activity, and GS-X pump activity were regulated separately to examine effects of the regulation on cisplatin cytotoxicity and cisplatin-induced DNA interstrand cross-links (ICL) in HepG2 cells. Seventy-percent depletion of GSH by buthionine sulfoximine (BSO) and 50% increase of GSH by monoethyl GSH ester (GSHe) potentiated and decreased cisplatin cytotoxicity, respectively. This was reflected by a significant decrease and increase of their respective IC(50) values by 62 and 107%. Cisplatin-induced ICL was also potentiated by depletion of GSH by BSO and decreased by enrichment of GSH by GSHe, as shown by a 125% increase and a 34% decrease of cross-linked DNA compared with control samples exposed to cisplatin alone (p = 0.008 and 0.03, respectively). On the other hand, inhibition of GST and GS-X pump by ethacrynic acid, quercetin, tannic acid, and indomethacin at concentrations that inhibited activities of GST and GS-X pump by more than 50% had no significant effects on cisplatin cytotoxicity and cisplatin-induced DNA ICL in these cells. The results showed that of the parameters measured, intracellular GSH seems to be the rate-limiting factor, and its regulation would provide a more promising strategy for sensitization of human liver tumor cells to cisplatin.

Inhibition of epidermal growth factor receptor tyrosine kinase by chalcone derivatives.

In our previous study, butein, a chalcone derivative, was found to be an inhibitor of tyrosine kinases and the inhibition was ATP-competitive. In this work, chalcone and seven chalcone derivatives were used to analyse the relationship between the structure of these compounds and their inhibition of tyrosine kinase activity. Three of chalcone derivatives, including butein, marein and phloretin, were found to have an ability to inhibit the tyrosine kinase activity of epidermal growth factor receptor (EGFR) in vitro. IC(50) was 8 microM for butein, 19 microM for marein and 25 microM for phloretin. The structural characterisations of these inhibitors suggest that the hydroxylations at C4 and C4' of these molecules may be required for them to act as EGFR tyrosine kinase inhibitors. The inhibition of EGF-induced EGFR tyrosine phosphorylation by butein was also observed in human hepatocellular carcinoma HepG2 cells, while marein and phloretin were inactive at the doses tested. Molecular modelling suggests that butein, marein and phloretin can be docked into the ATP binding pocket of EGFR. Hydrogen bonds and hydrophobic interaction appear to be important in the binding of these inhibitors to EGFR.

Glutathione-related factors are not correlated with sensitivity of human tumour cells to actinomycin D.

Glutathione (GSH) contents and activities of glutathione S-transferases (GST), glutathione reductase (GSH-RD), glutathione peroxidase (GSHpx) and glutathione conjugate export pump (GS-X pump) were determined in eight human tumour cell lines with different sensitivities to melphalan, a substrate of glutathione conjugation, and actinomycin D which has not been shown to be detoxified by glutathione-related mechanisms. Chang liver cells with highest GSH content and highest activities of GST, GSH-RD, GSHpx and GS-X pump were found to be most resistant to melphalan. Statistical analysis showed significant correlations between sensitivities of the human tumour cells to melphalan and the glutathione-related factors (r = 0.72-0.79; except for GST, r = 0.65, P = 0.08), while there were no significant correlations observed between sensitivities of the human tumour cells to actinomycin D and all the glutathione-related factors tested (r = -0.25-0.14). Significant correlations of the glutathione-related factors to resistance of human tumour cells to melphalan, a substrate of glutathione conjugation, but not to resistance of the human tumour cells to actinomycin D which has not been shown to be detoxified by glutathione-related mechanisms suggested that glutathione-related mechanisms contribute to drug resistance by increased detoxification of the drugs involved.

Quercetin-induced apoptosis in the monoblastoid cell line U937 in vitro and the regulation of heat shock proteins expression.

Quercetin, a widely distributed plant flavonoid, inhibits the growth of tumor cells and the expression of heat shock proteins. In this study, we examined quercetin-induced apoptosis of U937 cells in vitro and the regulation of heat shock proteins expression. The quercetin-induced apoptosis was demonstrated to be dose- and time-dependent. Flow cytometry analysis revealed that sub-G1 peak appeared at 12 hours, 24 hours and 48 hours respectively when treated with 50 mM of quercetin and at 6 hours, 12 hours, 24 hours and 48 hours with 100 mM of quercetin. Agarose gel electrophoresis demonstrated a typical ladder-like pattern of DNA fragments in the same time intervals with 50 mM of quercetin and at 6 hours only with 100 mM of quercetin. The distribution of DNA contents showed that U937 cells were accumulated mainly at G2/M phase when incubated with 50 mM of quercetin for 24 hours and 48 hours. There was no specific effect on cell cycle distribution at 100 mM of quercetin. On exposure to 100 mM of quercetin, the inhibition of hsp27 and hsp70 expression was observed from 12 hours to 48 hours and 6 hours to 48 hours respectively. The inhibition of hsp90 expression was not found eiyher at 50 mM or 100 mM of quercetin exposure. When incubated with 50 mM of quercetin, there was no inhibitory effect on hsp27, hsp70 and hsp90 expression by the Western blot analysis. These results suggested that it is dependent on the dose and exposure time of quercetin to induce apoptosis of U937 cells in vitro, to affect the cell cycle distribution and to regulate the expression of hsp27 and hsp70.

Induction of neuronal and inducible nitric oxide synthase in the motoneurons of spinal cord following transient abdominal aorta occlusion in rats.

BACKGROUND: Motoneurons in the spinal cord are especially vulnerable to ischemic injury and selectively destroyed after transient ischemia. Nitric oxide (NO) has been implicated in both neurodegneration and neuroprotection to ischemic insult. To evaluate the role of NO in pathophysiology to spinal cord ischemia, the expression of neuronal and inducible nitric oxide synthase (n-NOS and i-NOS) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in the motoneurons of the lumbosacral spinal cord was examined in a rat model with transient abdominal aorta (TAA) occlusion. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into sham-operated (n = 12) and TAA occlusion (n = 24) groups. TAA occlusion was induced by placement of a microvascular clamp around the abdominal aorta for 20 min. Three sham-operated and six TAA occlusion animals were sacrificed at each time interval at 4, 24, and 48 h and 7 days after operation. Tissue sections obtained from the lumbosacral spinal cord were processed for n-NOS, i-NOS, NADPH-d, and hematoxylin-eosin (HE) staining. Histological changes of motoneurons in ventral horn were assessed by HE staining. RESULTS: In sham-operated control animals, n-NOS-, i-NOS-, and NADPH-d-positive neurons were barely detectable in the ventral horn of the spinal cord. At 4 h after TTA occlusion, n-NOS and NADPH-d expression became evident in the motoneurons and was markedly enhanced at 24 and 48 h. i-NOS expression was also induced in the ventral horn motoneurons of the lumbosacral spinal cord at the same time points. Enzymatic expression in the motoneurons was diminished 7 days after operation. Hyperchromatic neurons indicative of cell death were observed in HE-stained specimens 7 days following TAA occlusion. CONCLUSIONS: The rapid induction of n-NOS, i-NOS, and NADPH-d in the motoneurons of ventral horn suggests that NO may be involved in the selective and delayed neuronal death in the spinal cord to the ischemic insult.

Daphnetin, one of coumarin derivatives, is a protein kinase inhibitor.

Protein kinases play key roles in the control of cell proliferation, differentiation and metabolism. In this work, we studied the effect of coumarin and its derivatives, including daphnetin, esculin, 2-OH-coumarin, 4-OH-coumarin and 7-OH-coumarin, on the activity of protein kinases. It was found that, in these compounds, only daphnetin was a protein kinase inhibitor. This compound inhibited tyrosine-specific protein kinase, EGF receptor (IC(50) = 7.67 microM), and serine/threonine-specific protein kinases, including cAMP-dependent protein kinase (PKA) (IC(50) = 9.33 microM) and protein kinase C (PKC) (IC(50) = 25.01 microM) in vitro. The inhibition of EGF receptor tyrosine kinase by daphnetin was competitive to ATP and non-competitive to the peptide substrate. The inhibition of EGF-induced tyrosine phosphorylation of EGF receptor by daphnetin was not observed in human hepatocellular carcinoma HepG2 cells. The structural comparison of daphnetin with coumarin and other coumarin derivatives suggests that the hydroxylation at C8 may be required for daphnetin acting as a protein kinase inhibitor.

GSH, GSH-related enzymes and GS-X pump in relation to sensitivity of human tumor cell lines to chlorambucil and adriamycin.

Glutathione (GSH) contents and activities of glutathione S-transferase (GST), glutathione reductase (GSH-RD), glutathione peroxidase (GSHpx) and glutathione conjugate export pump (GS-X pump) were determined in eight human tumor cell lines with different sensitivities to adriamycin and chlorambucil. Correlations between sensitivities of the human tumor cells to adriamycin and chlorambucil and the glutathione related factors were analyzed statistically. Sensitivities of the human tumor cells to chlorambucil were found to be correlated to all the glutathione related factors tested (r=0.68-0.88). IC50 values of adriamycin were also positively correlated to GSH contents and activities of GSH-RD, GSHpx and GS-X pump with r values ranging from 0.66 to 0.77 but not to GST activity (r=0.25). Chang liver cells with highest GSH content and highest activities of GST, GSH-RD, GSHpx and GS-X pump were most resistant to both adriamycin and chlorambucil. These data suggested that glutathione related factors may work as an overall detoxification system participating in the detoxification of anticancer drugs such as adriamycin and chlorambucil, and to be involved in cellular resistance to these drugs.

Butein, a specific protein tyrosine kinase inhibitor.

Butein, a plant polyphenol, was shown to be a specific protein tyrosine kinase inhibitor. This compound inhibited not only the epidermal growth factor (EGF)-stimulated auto-phosphotyrosine level of EGF receptor in HepG2 cells but also tyrosine-specific protein kinase activities of EGF receptor (IC50 = 65 microM) and p60c-src (IC50 = 65 microM) in vitro. The inhibition was competitive to ATP and non-competitive to the phosphate acceptor, poly (Glu, Ala, Tyr) 6:3:1 for EGF receptor tyrosine kinase. In contrast, butein non-significantly inhibited the activities of serine- and threonine-specific protein kinases, such as protein kinase C (PKC) and cAMP-dependent protein kinase (PKA).

Inhibition of glutathione reductase by plant polyphenols.

The effects of forty-one plant polyphenols on the activity of glutathione reductase (GSH-RD) were studied. These polyphenols showed varying degrees of concentration-dependent inhibition on the enzyme, with IC50 values that varied from approximately 40 microM to 1 mM. 4'-Hydroxychalcone and tannic acid were among the more potent inhibitors, with IC50 values of 47.3 and 50.4 microM, respectively. Different classes of polyphenols varied in potency in the following order: chalcones > tannic acid > flavonoids > coumarins > catechins. Analysis of structure-activity relationships showed certain chemical structures to be important for the inhibition of GSH-RD: (a) C-5 and C-7 hydroxylations in the A-ring, a carbonyl group at C-4, and the B-ring attached to C-2 in flavonoids; (b) C-2' and C-4' hydroxylations in chalcones; and (c) C-6 and C-7 hydroxylations in coumarins. The inhibition of GSH-RD by tannic acid and quercetin was time dependent and irreversible, whereas that by 4'-hydroxychalcone and esculin was reversible but not time dependent. Enhanced inhibition of GSH-RD by the four polyphenols 4'-hydroxychalcone, quercetin, butein, and acacetin was observed in the presence of NADPH. Kinetic studies showed that both tannic acid and 4'-hydroxychalcone exhibited non-competitive inhibition on GSH-RD towards glutathione disulfide.

Norcantharidin inhibits growth of human HepG2 cell-transplanted tumor in nude mice and prolongs host survival.

In this study, norcantharidin was compared with adriamycin and mitomycin C for its inhibitory action in the growth of cultured human hepatocellular carcinoma HepG2 cells. The IC50 of adriamycin and mitomycin C on HepG2 cells was 7.3 microM and 27 microM, respectively, whereas the IC50 of norcantharidin for inhibiting the growth of HepG2 cells was as high as 1900 microM. After HepG2 tumor-bearing nude mice were treated with 12 daily intraperitoneal injections of norcantharidin (2 mg/kg), the increase in tumor size was significantly slower than that of untreated controls. The mean survival time of untreated tumor-bearing nude mice was 129 days, whereas in the tumor-bearing nude mice treated with norcantharidin, the mean survival time was significantly prolonged to 194 days (P < 0.0001). It is concluded that norcantharidin may have a potential role in the treatment of human hepatocellular carcinoma.

Transmodulation of EGF receptor by interferon-gamma in human hepatocellular carcinoma HepG2 cells.

EGF receptor has been recognized to playing an important role in the regulation of normal and tumor cell growth. In this study, the transmodulatory effect of IFN gamma on EGF receptor of human hepatocellular carcinoma cell line HepG2 was investigated. The results demonstrated that IFN gamma was able to modulate EGF receptor of HepG2 cells by enhancing the tyrosine phosphorylation of the receptor. However, the effect appeared only if EGF was present, but not mediated by IFN gamma alone. No significant alteration was found in terms of the expression or the affinity of EGF receptor when HepG2 cells were treated with IFN gamma. In addition, EGF internalization in the cells was also not affected. Because IFN gamma is an inhibitory agent for the growth of HepG2 cells, this transmodulatory effect of IFN gamma on the tyrosine phosphorylation of EGF receptor might be associated with the inhibition of the growth of HepG2 cells.

Modulation of EGF receptor by tumor necrosis factor-alpha in human hepatocellular carcinoma HepG2 cells.

TNF alpha is known to exert multi-regulatory effects on normal and malignant cell functions by binding to the corresponding cell surface receptor. However, the existence of cross interaction between TNF alpha and EGF receptor has been proposed. In this study, we investigated the modulatory effect of TNF alpha on EGF receptor of a human hepatocellular carcinoma cell line-HepG2. The results suggested that TNF alpha was able to modulate the EGF receptor of HepG2 cells. The modulatory effect of TNF alpha on the EGF receptor of HepG2 cells exhibited its unique characteristics in comparison with the previous reports on other tumor cells. TNF alpha could also enhance the tyrosine phosphorylation of the EGF receptor of HepG2 cells. However, the effect appeared only if EGF was present, and was not mediated by TNF alpha alone. Therefore, the effect of TNF alpha on EGF receptor tyrosine phosphorylation in HepG2 cells was due to enhancing the receptor's response to EGF. TNF alpha was also able to reduce the affinity of the high-affinity receptor for EGF. However, there was no significant alteration in terms of the expression of EGF receptor, EGF internalization, or EGF degradation when HepG2 cells were treated with TNF alpha. Since TNF alpha is an inhibitory agent for HepG2 cell growth, this cross interaction between TNF alpha and EGF receptor may play a role in the inhibition of cell growth.

Genistein, a tyrosine kinase inhibitor, reduces EGF-induced EGF receptor internalization and degradation in human hepatoma HepG2 cells.

In this work, using the ECL Western blotting assay system, it was found that genistein, a specific tyrosine kinase inhibitor, was able to inhibit EGF-induced EGF receptor degradation and tyrosine phosphorylation in human hepatoma HepG2 cells. This inhibition was increased with increasing genistein concentration. With treatment of HepG2 cells with genistein at 37 degrees C for 30 min, the amount of internalized EGF, which was measured by the detection of the sorting of 125I-EGF in the cells, was remarkably decreased. Under the same conditions, in cells untreated with genistein, the degradation of EGF was significantly increased. After preincubation of HepG2 cells with and without genistein for 120 min at 37 degrees C, the ratio between degraded and released EGF was 16 and 24, respectively. These results suggest that EGF-induced internalization and degradation of EGF-EGF receptor complexes in HepG2 cells depend on EGF receptor tyrosine kinase activity.

Detection of EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells.

In this work, a simple, sensitive, and non-isotopic assay system for the detection of EGF-induced EGF receptor degradation and tyrosine phosphorylation in intact cells is described. In this system, boiling Laemmli sample buffer was directly added to cultured Chang liver cells to stop the reactions in the cells stimulated by EGF and to make whole-cell extracts. The effects of EGF concentration and incubation time on the EGF-induced degradation and tyrosine phosphorylation of EGF receptor were successfully determined using monoclonal anti-EGF receptor, recombinant anti-phosphotyrosine peroxidase conjugate, and enhanced chemiluminescence (ECL) Western blotting assay system. Unlike other assay systems, the use of radioisotopes was avoided in this determination. The assay system is linear up to 100 micrograms sample protein from whole-cell extracts for the detection of EGF receptor and EGF-induced autophosphorylation. This assay may be easily adopted for identification of other growth factor receptors and phosphotyrosine-containing proteins in intact cells, using appropriate anti-growth factor receptor antibodies.

EGF receptor in human Chang liver and hepatoma HepG2 cells.

Epidermal growth factor (EGF) receptor was detected in the human Chang liver and human hepatoma HepG2 cells. Both cell lines were found to be able to bind EGF. The expression of EGF receptor in Chang liver and HepG2 cells was 1.3 x 10(5) EGF receptors/cell and 1.8 x 10(5) EGF receptors/cell, respectively. In both cells, this receptor was identified by ECL Western blotting using monoclonal anti-EGF receptor antibody and by immunohistochemical assay using polyclonal anti-EGF receptor antibody. Some of internalized EGF was recycled in Chang liver cells, but not in HepG2 cells.

Comparison of the sensitivity of different India inks staining of electro-blotted proteins on filter membranes.

The sensitivity of different brands of India ink for staining proteins blotted onto filter membranes is described. Proteins that are electro-blotted onto filter membranes show better retention than those dot-blotted alone. Higgins engrossing waterproof black ink No. 893 can detect protein at a concentration as low as 5 ng which is much more sensitive than Coomassie blue. The nitrocellulose membrane from S&S is ideal for blotting proteins and gives low levels of background staining. The sensitivity of protein staining is however, affected by the types of India ink, ink concentrations, staining times and membrane lots. India ink is also found to be useful for staining circulating immune complexes separated by SDS-PAGE and electro-blotted onto filter membranes.

Studies of epidermal growth factor (EGF) receptor in plasma membrane from rat liver and hepatoma tissues.

Plasma membrane purified from Buffalo rat liver tissue not only had the ability to bind 125I-EGF (2.77 pg of EGF/mg of membrane protein), but also exhibited both high (Kd = 0.08 nM) and low (Kd = 5.67 nM) affinity receptors. However, the binding of EGF to hepatoma plasma membrane was insignificant. EGF receptor in plasma membrane from normal liver tissue was identified by ECL Western blotting using monoclonal anti-EGF receptor antibody and phosphorylated via the stimulation of EGF. This phosphorylation was inhibited by genistein, a tyrosine kinase inhibitor. However, these effects were not observed in hepatoma cell membrane. These results suggest that plasma membrane purified from normal rat liver tissue has a high level of functional EGF receptor, whereas, hepatoma plasma membrane lacks the receptor.