Increased lysine N-methylation of a 23-kDa protein during hepatic regeneration

The methylation of a 23-kDa nuclear protein increased after partial hepatectomy and methylation returned to basal levels after the initial stage of regeneration. The methylating enzyme was partially purified from rat liver by ammonium sulfate precipitation, DEAE-anion exchange chromatography and Butyl-Sepharose chromatography. The 23-kDa protein was purified from a nuclear fraction of liver tissue with SP-Sepharose. When the 23-kDa protein was methylated with the partially purified methyltransferase and analyzed on C(18) high performance liquid chromatography (HPLC), the methylated acceptor amino acid was monomethyl lysine (MML). Previously, only arginine N-methylation of specific substrate proteins has been reported during liver regeneration. However, in this report, we found that lysine N-methylation increased during early hepatic regeneration, suggesting that lysine N-methylation of the 23-kDa nuclear protein may play a functional role in hepatic regeneration. The methyltransferase did not methylate other proteins such as histones, hnRNPA1, or cytochrome C, suggesting the enzyme is a 23-kDa nuclear protein- specific lysine N-methyltransferase.     

Calcium-binding protein regucalcin stimulates the uptake of Ca2+ by rat liver mitochondria

The effect of the calcium-binding protein regucalcin on the Ca2+ transport system in rat liver mitochondria was investigated. Ca2+ transport was assayed by the method of Millipore filtration to estimate mitochondrial 45Ca2+ accumulation. 45Ca2+ uptake was stimulated by the presence of regucalcin (1.0 and 2.0 microM). This stimulation was remarkable during 1.0 min after 45Ca2+ addition, while appreciable stimulation was no longer seen at 3 min. Regucalcin (2.0 microM)-induced stimulation of 45Ca2+ uptake was prevented by the presence of ruthenium red (1.0 microM) and lanthanum chloride (0.1 mM). Regucalcin (2.0 microM) did not increase the mitochondrial adenosine triphosphatase (ATPase) activity during 3.0 min after Ca2+ addition. Meanwhile, 45Ca2+, which accumulated in the mitochondria during 5.0 min after 45Ca2+ addition, was not released by the addition of regucalcin. Regucalcin may stimulate Ca2+ uptake in rat liver mitochondria independently of the energy.     

Regucalcin-induced Ca2+ release from rat liver microsomes: the effect is inhibited by heparin

The effect of heparin on the calcium-binding protein regucalcin-stimulated Ca2+ release from rat liver microsomes was investigated. Ca2+ release was assayed by the method of Millipore filtration to estimate microsomal 45Ca2+ accumulation following the addition of 10 mM adenosine triphosphate. The addition of regucalcin (1.0 microM) or inositol 1,4,5-trisphosphate [Ins(1,4,5)P3; 1.0 microM] stimulated 45Ca2+ release from rat liver microsomes. These effects were completely inhibited by the presence of heparin (10.0 micrograms/ml). Regucalcin did not enhance the effect of Ins(1,4,5)P3. These results suggest that regucalcin affects 45Ca2+ release involved in Ins(1,4,5)P3 action in rat liver microsomes.     

Formation of a benzimidazole ring accompanied by the cleavage of a nitrogen-carbon bond

The reaction of thionyl chloride with arylamides of aromatic acids, which usually gives rise to stable imidoyl chlorides, leads in the case of N-(o-dimethylaminophenyl)benzimide and N-(o-piperidinophenyl)-benzimide to the formation of a benzimidazole ring, which is accompanied by the cleavage of the nitrogen-carbon bond. This reaction and the reaction of chloral oxime with o-dialkylaminoanilines studied previously, which also leads to the formation of a benzimidazole ring, are compared with the Braun reaction. It has been shown that when thionyl chloride reacts with N-(o-dimethylaminophenyl)isonitrosoacetamide, no formation of a benzimidazole ring takes place and only N-(o-dimethylaminophenyl)carbamoyl cyanide is formed.     

Phosphate-catalyzed degradation of D-glucosone in aqueous solution is accompanied by C1-C2 transposition

Pathways in the degradation of the C(6) 1,2-dicarbonyl sugar (osone) D-glucosone 2 (D-arabino-hexos-2-ulose) in aqueous phosphate buffer at pH 7.5 and 37 °C have been investigated by (13)C and (1)H NMR spectroscopy with the use of singly and doubly (13)C-labeled isotopomers of 2. Unlike its 3-deoxy analogue, 3-deoxy-D-glucosone (3-deoxy-D-erythro-hexos-2-ulose) (1), 2 does not degrade via a 1,2-hydrogen shift mechanism but instead initially undergoes C1-C2 bond cleavage to yield d-ribulose 3 and formate. The latter bond cleavage occurs via a 1,3-dicarbonyl intermediate initially produced by enolization at C3 of 2. However, a careful monitoring of the fates of the sketetal carbons of 2 during its conversion to 3 revealed unexpectedly that C1-C2 bond cleavage is accompanied by C1-C2 transposition in about 1 out of every 10 transformations. Furthermore, the degradation of 2 is catalyzed by inorganic phosphate (P(i)), and by the P(i)-surrogate, arsenate. C1-C2 transposition was also observed during the degradation of the C(5) osone, D-xylosone (D-threo-pentose-2-ulose), showing that this transposition may be a common feature in the breakdown of 1,2-dicarbonyl sugars bearing an hydroxyl group at C3. Mechanisms involving the reversible formation of phosphate adducts to 2 are proposed to explain the mode of P(i) catalysis and the C1-C2 transposition. These findings suggest that the breakdown of 2 in vivo is probably catalyzed by P(i) and likely involves C1-C2 transposition.     

Effect of protein binding on the disposition of cephalexin and cefazolin in a simultaneous perfusion system of rat liver and kidney

The effect of protein binding on the disposition of cephalexin (CEX) and cofazolin (CEZ) was investigated in a simultaneous perfusion system of rat liver and kidney. In the present study, we used bovine serum albumin (BSA) or human serum albumin (HSA) as plasma protein to control the degree of perfusate protein binding of drugs. Total clearance (CLt) of CEX perfused with BSA (0.70 +/- 0.27 ml/min) was slightly smaller than that with HSA (0.89 +/- 0.08 ml/min), corresponding to the unbound fraction of the drug in the perfusate plasma. On the other hand, CLt of CEZ perfused with BSA (0.90 +/- 0.20 ml/min) was significantly larger than that with HSA (0.32 +/- 0.10 ml/min). The unbound fraction of CEZ to BSA (0.703 +/- 0.052) was much larger than that to HSA (0.253 +/- 0.017) and the clearance of the unbound drug did not differ significantly between two kinds of albumin perfusate (1.30 +/- 0.40 ml/min for BSA and 1.26 +/- 0.40 ml/min for HSA). These results suggest that plasma protein binding is an important factor determining the biliary clearance as well as the urinary clearance of drugs.     

Determination of the distance between the two neutral flavin radicals in augmenter of liver regeneration by pulsed ELDOR

Pulsed electron-electron double resonance (ELDOR) has been used to obtain structural information from a FAD-dependent sulfhydryl oxidase, Augmenter of Liver Regeneration (ALR). ALR is a homodimer with each subunit containing a noncovalently bound FAD cofactor. Both FADs may be converted into the blue neutral radical form by aerobic treatment with DTT. From three-pulse and four-pulse ELDOR experiments, a distance of 26.1 +/- 0.8 A could be determined between the FAD cofactors in human ALR. Taking into account the electron spin density distribution in a neutral flavin radical obtained from density functional theory calculations, a distance of 26.9 A could be estimated for the separation of the spin centers in the X-ray structure of rat ALR. The good agreement confirms that rat ALR may be used as a model for mechanistic discussions of human ALR. The experiments also demonstrate that neutral flavin radicals have the appropriate properties to be used as intrinsic spin labels for distance determinations in proteins.     

The stimulation of the cholesterol synthesis in rat liver by hydrocortisone.

A single dose of 1 mg hydrocortisone per rat stimulates the incorporation of labeled acetate into the cholesterol of the liver by a factor of 2.7 measured 18 hours after the administration of the hormone and 2 hours after the tracer dose of labeled acetate.     

The mitogen-activated protein kinase phosphatase-1 (MKP-1) gene is a potential methylation biomarker for malignancy of breast cancer

The mitogen-activated protein kinase (MAPK) phosphatase- 1 (MKP-1) belongs to the MAPK cascades which are central to cell proliferation and apoptosis. The carcinogenic role of MKP-1 has been reported in many types of cancer but it has rarely been investigated in breast cancer. The present study was designed to evaluate the MKP-1 mRNA expression and its possible regulation by methylation of MKP-1 promoter in the model of several breast cancer cell lines and tissues as well as controls. Our data demonstrate MKP-1 mRNA expression significantly decreased in five breast cancer cell lines compared to breast controls (P<0.01). Using the methylation-specific PCR (MSP) analysis, the unmethylated reaction (U) is dominant in both normal cell lines and benign breast tumors (100% vs. 86.2%), whereas the methylated reaction (M) is dominant in both breast cancer cell lines and invasive breast tumors (100% vs. 57.2%). In terms of methylation ratio (M/M+U), methylation level in MKP-1 promoter is significantly higher in the invasive breast tumor tissues (n = 152) than in benign breast tumor tissues (n = 29) (P<0.0001). Assessing the methylation ratio of the promoter of MKP-1 gene to diagnose the breast malignancy (invasive vs. benign), the area under the receiver- operating characteristic (ROC) curve was 0.809 (95% CI: 0.711-0.906, P<0.001). The best performance for this prediction has a sensitivity of 76.32% and a specificity of 82.76% at the cutoff value of 0.38. Taken together, we firstly demonstrated that the promoter methylation of MKP-1 gene is a potential breast cancer biomarker for breast malignancy.     

Kinetics of catalyst regeneration by coke combustion. I. Increased reaction rate due to the presence of chromium

A kinetic study has been carried out of the regeneration of a commercial Cr₂O₃/Al₂O₃ catalyst by coke combustion. The results obtained show that the reaction rate is several orders of magnitude higher than that obtained on catalysts without transition metals.

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