Acetylation is essential for nuclear heme oxygenase-1-enhanced tumor growth and invasiveness.

Overexpression of heme oxygenase-1 (HO-1), an endoplasmic reticulum-anchored enzyme, is observed in many cancers. HO-1 nuclear translocation has been shown to correlate with progression of several cancers. We recently reported that HO-1 is susceptible to intramembrane proteolysis and translocates to the nucleus to promote cancer growth and invasiveness without depending on its enzymatic activity. In the present study, we show that the HO-1 lacking C-terminal transmembrane segment (t-HO-1) was susceptible to acetylation by p300 and CREB-binding protein (CBP) histone acetyltransferase in the nucleus. Mass spectrometry analysis of HO-1 isolated from human embryonic kidney cells 293T (HEK293T) cells overexpressing CBP and t-HO-1 revealed two acetylation sites located at K243 and K256. Mutation of both lysine residues to arginine (R) abolished t-HO-1-enhanced tumor cell growth, migration and invasion. However, mutation of the lysine residues to glutamine (Q), a mimic of acetylated lysine, had no significant effect on t-HO-1-mediated tumorigenicity. Mechanistic studies demonstrated that transcriptional factor JunD interacted with wild-type (WT) t-HO-1 and mutant carrying K243/256Q but not K243/256 R mutation. Moreover, JunD-induced AP-1 transcriptional activity was significantly enhanced by coexpression with WT and acetylation-mimic but not acetylation-defective t-HO-1. Consistent with the in vitro observations, the implication of K243/256 acetylation in t-HO-1-enhanced tumorigenicity was also demonstrated in xenograft models. Immunohistochemistry performed with a specific antibody against acetyl-HO-1 showed the positive acetyl-HO-1 nuclear staining in human lung cancer tissues but not in the corresponding non-tumor tissues, supporting its clinical significance. Collectively, our findings highlight the importance of nuclear HO-1 post-translational modification in the induction of cancer progression.

γ-Synuclein Expression Is a Malignant Index in Oral Squamous Cell Carcinoma.

Dysregulation of γ-synuclein (SNCG) has been reported in many cancers; however, its role in cancer development is still controversial. Here, we examined the potential involvement of DNA methylation in regulating SNCG and its role in oral squamous cell carcinoma (OSCC). We used 8 OSCC cell lines to investigate SNCG methylation and expression. SNCG methylation was examination by methylation-specific polymerase chain reaction and bisulfate sequencing. Cells showing a high degree of SNCG methylation were treated with 5-aza (methylation inhibitor), and changes in their methylation and expression profiles were analyzed. Functional effects of SNCG in OSCC were examined by its overexpression and knockdown. Additionally, methylation and expression of SNCG in OSCC tissues were investigated and correlated with clinicopathologic features. All OSCC cells showed detectable SNCG expression at the mRNA and protein levels. Methylation-specific polymerase chain reaction and bisulfate sequencing revealed high SNCG expression in SCC25 cells with the unmethylated allele, and their 15 CpG islands were unmethylated. The methylated allele was detected only in OEC-M1 cells exhibiting low SNCG expression, and their CpG islands were partially methylated. 5-aza treatment in OEC-M1 cells attenuated methylation and restored SNCG expression. SNCG overexpression increased colony forming, migration, and invasion abilities in OEC-M1 cells. Silencing SNCG in SCC25 cells suppressed these behaviors. All 25 tumor-adjacent normal tissues were negative for SNCG immunostaining. SNCG upregulation was frequently observed in dysplastic and OSCC tissues. Positive SNCG expression was found in 45% (37 of 82) OSCC tissues. Positive SNCG expression in OSCC significantly correlated with cancer staging and lymph node metastasis. However, SNCG methylation did not correlate with its expression and clinicopathologic variables in OSCC tissues. DNA methylation may participate in regulating SNCG expression in some OSCC cells. SNCG upregulation could be involved in OSCC progression.

Signal peptide peptidase-mediated nuclear localization of heme oxygenase-1 promotes cancer cell proliferation and invasion independent of its enzymatic activity.

Heme oxygenase-1 (HO-1) is a heme-degrading enzyme anchored in the endoplasmic reticulum by a carboxyl-terminal transmembrane segment (TMS). HO-1 is highly expressed in various cancers and its nuclear localization is associated with the progression of some cancers. Nevertheless, the mechanism underlying HO-1 nuclear translocation and its pathological significance remain elusive. Here we show that the signal peptide peptidase (SPP) catalyzes the intramembrane cleavage of HO-1. Coexpression of HO-1 with wild-type SPP, but not a dominant-negative SPP, promoted the nuclear localization of HO-1 in cells. Mass spectrometry analysis of cytosolic HO-1 isolated from HeLa cells overexpressing HO-1 and SPP revealed two adjacent intramembrane cleavage sites located after S275 and F276 within the TMS. Mutations of S275F276 to A275L276 significantly hindered SPP-mediated HO-1 cleavage and nuclear localization. Nuclear HO-1 was detected in A549 and DU145 cancer cell lines expressing high levels of endogenous HO-1 and SPP. SPP knockdown or inhibition significantly reduced nuclear HO-1 localization in A549 and DU145 cells. The positive nuclear HO-1 stain was also evident in lung cancer tissues expressing high levels of HO-1 and SPP. Overexpression of a truncated HO-1 (t-HO-1) lacking the TMS in HeLa and H1299 cells promoted cell proliferation and migration/invasion. The effect of t-HO-1 was not affected by a mutation in the catalytic site. However, blockade of t-HO-1 nuclear localization abolished t-HO-1-mediated effect. The tumorigenic effect of t-HO-1 was also demonstrated in the mouse model. These findings disclose that SPP-mediated intramembrane cleavage of HO-1 promotes HO-1 nuclear localization and cancer progression independent of HO-1 enzymatic activity.

Lipoprotein, lecithin:cholesterol acyl transferase and acetyl CoA carboxylase in stroke-prone spontaneously hypertensive rats fed a diet high in eicosapentaenoic acid.

To investigate the effect of dietary eicosapentaenoic acid (EPA) on plasma lipoprotein levels, lecithin:cholesterol acyltransferase (LCAT) activity and liver acetyl CoA carboxylase activity, highly concentrated EPA (78%) purified from sardine oil was fed to stroke-prone spontaneously hypertensive rats (SHRSP) for 30 days. Significantly (P < 0.05) lower systolic blood pressure and plasma total cholesterol were observed in rats fed an EPA diet. In addition, higher HDL cholesterol and lower VLDL cholesterol levels were found in rats fed the EPA diet as compared with rats fed the control diet. However, no significant change of plasma LDL cholesterol was observed in rats between the two dietary groups. EPA supplementation increased the activity of plasma LCAT in rats. In addition, rats fed an EPA diet had lower liver total lipids and adipose tissue weights. However, higher liver acetyl CoA carboxylase activity was observed in rats fed the EPA diet. Results from the present study suggest that dietary EPA might stimulate the plasma lipoprotein metabolism and also alter lipogenesis in the liver of SHRSP rats.

Serum adenosine deaminase in the early diagnosis of typhoid fever.

To study the usefulness of the enzyme adenosine deaminase for the early diagnosis of typhoid fever, its activity in serum was assayed in 277 children admitted to the Hospital Guillermo Grant Benavente at Concepción, Chile, from March, 1988, to December, 1990. The children were distributed into seven groups: control, N = 82; bacteremia, N = 8; acute viral respiratory infection, N = 43; febrile children with miscellaneous etiologies, N = 49; pulmonary tuberculosis, N = 3; hepatitis A virus infection, N = 30; and typhoid fever, N = 62. The medium serum adenosine deaminase values were significantly higher in children with typhoid fever (P < 0.0001) in relation to the values in the control group (122.2 +/- 40.7 vs 28.1 +/- 8.4 units/liter at 37 degrees C). This test had a sensitivity of 91.9% and a specificity of 92.5% in identifying the patient with typhoid fever when using 80 units/liter as the cutoff values. The positive predictive value of the test was 83.8% and the negative predictive value was 96%. Determination of adenosine deaminase values in serum could be helpful in the early diagnosis of typhoid fever.

Tris(2,2'-bipyridyl)ruthenium(III)-based electrochemiluminescence detector with indium/tin oxide working electrode for capillary electrophoresis.

A novel tris(2,2'-bipyridyl)ruthenium(III) [Ru(bpy)3(3+)]-based electrochemiluminescence (ECL) detector for capillary electrophoresis (CE) has been developed. The detector was of the wall-jet configuration and an indium/tin oxide (ITO)-coated glass plate was used as the working electrode for end-column detection. Potential control of the ITO electrode was provided using a DC battery, without decoupling the detector from the CE field. Electrochemical behavior of Ru(bpy)3(2+) at the ITO electrode was found to be analogous to that at a Pt electrode. In the presence of tertiary or some secondary amines, ECL emission due to reaction between in situ generated Ru(bpy)3(3+) and analytes can be observed at the ITO surface. With 15 mM sodium borate (pH 9.5) plus 1 mM Ru(bpy)3(2+) present in the detection cell and the ITO electrode biased at 1.5 V (vs. Pt wire reference), a detection limit of 1 microM proline with a theoretical plate number of 4000 was obtained using the developed CE-ECL detection system. The detector response was found to be analyte-dependent, e.g. tryptophan gives no response, and the response for histidine is about 13-fold lower than that of proline.

Analysis of baclofen by capillary electrophoresis with laser-induced fluorescence detection.

A new analytical method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. Naphthalene-2,3-dicarboxaldehyde was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) and a He-Cd laser (excitation at 442 nm, emission at 500 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the concentration limit of detection was in the nanomolar level. Coupled with a simple cleanup procedure, the method was successfully applied to the analysis of baclofen in human plasma. Recovery of spiked baclofen in plasma was 98%. The relative standard deviation values on peak size and migration time were 7.9% and 0.4%, respectively. The limit of detection of baclofen in plasma was 10 ng/ml.

Toxic effects of grass carp, snake and chicken bile juices in rats.

To evaluate the toxic effects of different animal bile juices, male Long-Evans rats were used and treated orally with different doses (0.03-0.6 ml) of grass carp, snake and chicken bile juices. After treating with one high dose (0.6 ml) for 6 and 24 h, the levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine in the plasma of rats in the grass carp bile juice group became higher than those of the other two bile treated groups. After 3-days periodic treatment with 0.3 ml of each animal bile juice for 28 days, the levels of GOT, GPT, BUN and creatinine in the plasma of rats were significantly increased, especially the grass carp bile juice-treated rats. It appeared that the rats administered with snake and chicken bile juices for a much longer time were poisoned and had the same symptoms as those treated with grass carp bile juice.

Effects of dehulled adlay on the culture count of some microbiota and their metabolism in the gastrointestinal tract of rats.

Experiments were conducted to study the effect of a dietary supplement of dehulled adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) on the culture counts of some important groups of intestinal bacteria and their metabolism in the gastrointestinal (GI) tract of Sprague-Dawley rats. Rats were divided into four groups, and each group was fed a diet containing different levels of dehulled adlay for 30 days as follows: 0% (control), 5%, 20%, and 40%. All animals fed with adlay had normal healthy intestinal walls and no pathogenic signs whatsoever. There were no significant differences in body weight gain or the cecal pH between different groups of rats. Both the 20% and 40% groups had lower culture counts of enterics in their feces than the 5% and control groups, whereas the culture counts of fecal lactic acid bacteria were higher in feces of rats fed with adlay than in the control group. Cecal total short-chain fatty acid (SCFA) content and fecal SCFA were significantly higher in the 20% and 40% groups than in the control and 5% groups. All the adlay-fed rats had a higher fecal butyric acid concentration than the control rats. It is concluded that adlay has a significant influence on the growth of intestinal bacteria, which may ultimately affect the physiology and other functions of GI tracts of rats.

Effect of trans fatty acids on plasma lipids, platelet function and systolic blood pressure in stroke-prone spontaneously hypertensive rats.

To investigate the effect of trans fatty acids on plasma lipid levels and systolic blood pressure, hydrogenated corn oil was fed to SHRSP (stroke-prone spontaneously hypertensive rats) and WKY (Wistar-Kyoto) rats for 30 days. Significantly lower systolic blood pressure and plasma total cholesterol were observed in SHRSP rats fed trans fatty acids when compared with rats fed cis fatty acids from olive oil. In addition, higher HDL cholesterol and lower VLDL plus chylomicron cholesterol levels were found in SHRSP rats fed trans fatty acids. Although no significant changes of systolic blood pressure and plasma total cholesterol levels were observed in WKY rats after trans fatty acids treatment, WKY rats fed trans fatty acids had lower plasma LDL cholesterol and higher HDL cholesterol levels. In addition, platelet aggregation induced by collagen was decreased in WKY rats fed trans fatty acids. It is interesting that trans fatty acids increased the activity of plasma lecithin:cholesterol acyltransferase (LCAT) in both SHRSP and WKY rats. The observed influence of trans fatty acids on plasma lipid levels, systolic blood pressure and platelet aggregation suggests that trans fatty acids might prevent thrombotic disorders in SHRSP rats.

Effect of dietary protein on the peroxidation of eicosapentaenoic acid in stroke-prone spontaneously hypertensive rats.

In order to investigate the effect of dietary EPA on liver GSH peroxidase (GSH-Px) activity in rats, highly concentrated EPA (78% ethyl ester form) was administrated to SHRSP (Stroke-prone spontaneously hypertensive rat) that were fed a casein, SPI (soybean protein isolate) or SPI diet with methionine for 4 weeks. The content of liver GSH in rats fed SPI was lower than that of rats fed the casein diet. Although no significant difference of liver GSH-Px was observed in rats after EPA supplement, a decrease of liver GSH-Px activity was found in rats fed the SPI diet when compared with rats fed the casein diet. The changes of liver GSH content and GSH-Px activity in rats fed SPI were found to be associated with methionine supplement. Addition of methionine to the SPI diet resulted in an increase of liver GSH content and GSH-Px activity. In addition, liver lipid peroxide concentration was increased in rats fed the SPI diet after EPA treatment. In contrast, EPA administered rats fed the SPI diet containing methionine showed a lower liver lipid peroxide concentration. These results suggest that methionine may play an important role in regulation of the utilization of EPA in SHRSP when fed a SPI diet.

Effect of dietary fish oil on plasma lipoprotein cholesterol in rats fed a diet enriched in cholesterol and sucrose.

The aim of this study was to examine the effect of dietary fish oil on plasma lipoprotein cholesterol levels in rats fed different carbohydrate sources. Male Wistar rats fed a soy bean oil diet or a fish oil diet containing 0.5% cholesterol were studied for 7 weeks. Corn starch or sucrose were used as carbohydrate sources in the experimental diet. Fish oil supplementation significantly (p < 0.05) decreased plasma VLDL (very low density lipoprotein) cholesterol in rats fed a diet containing corn starch. However, there was no significant difference in plasma total cholesterol and LDL (low density lipoprotein) cholesterol in rats fed a corn starch diet with fish oil treatment. In the experiment with sucrose, significantly (p < 0.05) decreased plasma total cholesterol, LDL cholesterol and VLDL cholesterol were observed in rats fed a fish oil diet. Although higher plasma total and VLDL cholesterol levels were found in rats fed the sucrose diet when compared with those fed the corn starch diet, no significant difference between the corn starch group and the sucrose group was observed in rats after fish oil treatment. Results from the present study suggest that the carbohydrate source might play an important role in the regulation of plasma lipoprotein metabolism in rats fed fish oil.

Variation of plasma cholesterol levels in rats fed trans fatty acids or cis fatty acids.

To investigate the effect of dietary trans fatty acids on plasma and liver lipids, 16 Sprague Dawley male rats fed the hydrogenated soybean oil (Trans fat) or Cis fat from olive oil with two similar dietary fatty acid ratios for 9 weeks were studied. Higher plasma total cholesterol and LDL (low density lipoprotein) cholesterol levels were observed in rats fed trans fat diet when compared with rats fed the cis fat diet after 2 weeks of feeding. However, no significant changes in plasma total cholesterol and LDL cholesterol levels were found in rats of both dietary groups at 4-weeks of feeding. Rats fed trans fatty acids had lower plasma total cholesterol, LDL and VLDL (very low density lipoprotein) cholesterol levels at the end of the experimental period. Although significantly (p < 0.05) lower liver triacylglycerol contents were found in rats fed trans fat diet, no significant (p > 0.05) changes in liver cholesterol and phospholipids contents were observed in rats after trans fatty acids treatment. It is interesting that lower saturated to polyunsaturated ratios in fatty acid composition of plasma VLDL total lipids were found in rats fed trans fat diet. Results from this study suggest that the changes in plasma lipoprotein cholesterol in rats fed trans fatty acids might be related to the long or the short term study, and dietary trans fatty acids may alter the plasma lipoprotein metabolism in rats.

Effect of fish oil on plasma lipoproteins, liver glucose-6-phosphate dehydrogenase and glucose-6-phosphatase in rats.

To investigate the effect of dietary fish oil on the lipid and glucose metabolism, Wistar rats were fed lard (10% fat by weight) or fish oil (5% lard + 5% fish oil) diet for 7 weeks. Significantly (p < 0.05) lower plasma total cholesterol, low density lipoprotein cholesterol (VLDL-C) levels were observed in rats fed a diet containing fish oil when compared with rats fed lard diet. Fish oil supplementation decreased plasma lactic acid and free fatty acid levels. In addition, lower plasma lactate dehydrogenase (LDH) activity and an improved glucose tolerance ability were observed in rats fed fish oil diet. However, no significant change in plasma glucose level was found in rats between the two dietary groups. Although rats fed a diet containing fish oil had significantly (p < 0.05) decreased total cholesterol and phospholipid contents in the liver, there was no significant difference in liver triglyceride content between the two dietary groups. It is interesting to note that fish oil supplement significantly (p < 0.05) decreased liver glucose-6-phosphate dehydrogenase (G-6-PDH) and glucose-6-phosphatase (G-6-Pase) activities. Results from the present study suggest that dietary n-3 polyunsaturated fatty acids might play an important role in regulation of glucose and lipid metabolism in rats.

Effect of dietary eicosapentaenoic acid on plasma lipids and platelet function in stroke-prone spontaneously hypertensive rat.

An investigation was undertaken to study the effect of EPA in rats fed different protein sources. A highly concentrated EPA (78% EPA, ethyl ester form), manufactured from sardine oil was administered to SHRSP (Stroke-prone spontaneously hypertensive rat) and WKY (Wistar Kyoto) for 4 weeks. Casein or SPI (soy protein isolate) was used as protein source in the experimental diet. In the experiment concerning casein diet, showing significant decrease in systolic blood pressure, plasma lipids of SHRSP were observed after EPA treatment, but no significant difference was found in SPI diet group. Although there was no significant change in systolic blood pressure of WKY after EPA treatment, a similar effect of EPA on plasma lipids level and platelet aggregation were also observed in WKY. However, supplementing methionine to SPI diet induced the reducing effect of EPA in rats. In addition, higher level ratios of EPA to arachidonic acid were observed in the plasma and platelets of rats fed SPI diet containing methionine supplement when compared with rats fed SPI diet. It was suggested that the amino acid profile was related to the effective utilization of EPA in rats.

Plasma lipoprotein cholesterol levels in rats fed a diet enriched in cholesterol and cholic acid.

To investigate the effects of dietary cholesterol and cholic acid on plasma cholesterol levels, rats fed a cholesterol-free diet or a diet enriched in cholesterol (0.5% or 1%) with or without cholic acid supplementation were studied for 4 weeks. Although 0.5% cholesterol supplementation showed no effect on plasma total cholesterol and LDL-cholesterol levels in rats fed a diet without cholic acid treatment, the addition of dietary cholic acid caused an increase in plasma total cholesterol, LDL-cholesterol and VLDL-cholesterol levels in rats fed a cholesterol-rich diet. There was no significant change in HDL-cholesterol levels among the dietary groups. Rats fed a diet enriched in cholesterol have increased liver total lipids and total cholesterol contents. In addition, lower liver lipid peroxide concentration was found in rats fed a cholesterol-rich diet when compared with those fed the control diet. It is interesting that cholic acid supplementation led to an increase in hepatic cholesterol content and a decrease in liver lipid peroxide concentration in rats fed a cholesterol-rich diet. Results from this study suggest that dietary cholesterol and cholic acid might play an important role in regulation of lipid metabolism in rats.

The effect of adlay oil on plasma lipids, insulin and leptin in rat.

This study was designed to investigate the effect of dietary adlay oil on plasma lipids, insulin and lipid peroxidation levels in rats. Twenty-four male Wistar rats fed diet containing adlay oil and cholesterol were studied for 4 weeks. The animals were divided into three groups: (1) 10% lard (control) group; (2) 5% lard + 5% adlay oil (5% adlay oil) group; and (3) 10% adlay oil group. Although there was no significant difference in body weight at the end of the feeding study, rats fed a diet containing adlay oil showed a significant decrease in adipose tissue weight and relative adipose weight. In addition, the rats fed the adlay oil showed significantly decreased low-density lipoprotein cholesterol (LDL-C), insulin, leptin and thiobarbituric acid reactive substance (TBARS) concentrations after 4 weeks of the feeding study. Although a significant decrease in total plasma cholesterol was observed in rats fed the 5% adlay oil diet, no significant difference was observed between the 10% adlay oil and control groups, and neither was a significant difference in liver TBARS concentration found between the dietary groups. Results from this study suggest that dietary adlay oil can reduce leptin, adipose tissue and LDL-C levels in rats.

Chiral analysis of baclofen by alpha-cyclodextrin-modified capillary electrophoresis and laser-induced fluorescence detection.

An enantioselective method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoresis (CE) separation and laser-induced fluorescence (LIF) detection has been developed. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of the nonfluorescent drug. alpha-Cyclodextrin (alpha-CD) was included in the buffer as a chiral selector for the separation of NDA-labeled S-(+)- and R-(-)-baclofen. Optimal resolution and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) containing 7 mM alpha-CD and a He-Cd laser (lambda ex = 442 nm, lambda em = 500 nm). Combined with a simple cleanup procedure, this method can be applied to the analysis of baclofen enantiomers in human plasma. The relative standard deviation (RSD) values on peak areas of a plasma sample containing 1.0 microM racemic baclofen were 6.4 and 4.9% (n = 8) for the S-(+)- and R-(-)-enantiomer, respectively. The RSD value on migration times of both enantiomers was 0.5% (n = 8). Calibration graphs for S-(+)- and R-(-)-baclofen in plasma showed a good linearity (r > or = 0.999) in the concentration range of 0.1-2.0 microM. The limit of detection of baclofen in plasma was about 10 ng/mL.