Thiamine diphosphate pool and its biosynthesis in the liver in galactosamine hepatitis

The hepatitis-like changes were induced in the liver of albino female rats weighing 120-150 g and fed on the appropriate vivarium diet by single parenteral administration of hydrochloride galactosamine in a dose of 0.9 or 1.8 mmol per 1 kg of body weight. The thiamine diphosphate level in the cytosol fraction of the liver decreased 24 h after the preparation administration, the same in blood but with the higher dose used. The activity of pyruvate dehydrogenase, a thiamine diphosphate dependent enzyme, decreased similarly. The cytosol transketolase activity lowered by 38-39%. The coenzyme biosynthesis disturbance due to a fall by 49-58% in the thiamine pyrophosphatase activity is considered to be responsible for hydrochloride galactosamine-induced decrease in the thiamine diphosphate pool. Specificity of the thiamine diphosphate pool disturbance and discoordination of thiamine diphosphate dependent enzymes in the liver are observed under administration of hydrochloride galactosamine.     

On the pathogenesis of galactosamine hepatitis. Indications of extrahepatocellular mechanisms responsible for liver cell death.

In order to evaluate the pathogenesis of galactosamine hepatitis, the action of galactosamine on mast cells, and alteration in the complement system suring the course of this experimental injury were studied. It has been previously demonstrated that rat livers after colectomy are refractory to galactosamine-induced liver cell necrosis and inflammation. For this reason colectomized animals were used to see whether the biochemical alterations produced by this aminosugar and thought to be responsible for cell death developed. Results showed: 1. galactosamine potently degranulates mast cells in vivo and in vitro, 2. the complement system is a) activated during the course of galactosamine hepatitis, probably by circulating endotoxins, and b) is essential for liver cell death in galactosamine hepatitis, and 3. colectomy does not prevent biochemical changes known to occur during galactosamine metabolism. It is concluded that death of galactosamine-injured liver cells is triggered by extrahepatocellular mechanisms, which lead ultimately to an activated complement system by endotoxins. It is postulated that related mechanism may also occur in viral hepatitis and in fulminant hepatic failure in man.     

Orotate uptake and metabolism in normal and neoplastic tissues

The uptake of [3H]orotate was greater in mouse liver than in hepatoma but the difference was less marked than in the rat. Of the tissues examined, a high uptake of [3H]orotate was restricted to the liver and kidney in rat, mouse and guinea-pig. We confirmed that a high orotate diet greatly increases the ratio of UTP to ATP concentration in rat liver but we observed that there is little change of this nucleotide ratio in kidney. Evidence was obtained for a different pattern of orotate metabolism in rat liver and kidney.     

Orotate phosphoribosyltransferase from Corynebacterium ammoniagenes lacking a conserved lysine

The pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting from Corynebacterium ammoniagenes ATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 in Salmonella enterica serovar Typhimurium OPRTase) in the C. ammoniagenes OPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinant Escherichia coli strain. The molecular mass of the purified OPRTase was determined to be 45.4 ± 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 ± 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn²⁺ and Co²⁺. The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. The K_m values for the four substrates were determined to be 33 μM for orotate, 64 μM for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 μM for orotidine-5-phosphate (OMP), and 36 μM for pyrophosphate. The K_m value for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.     

Transition State Analogues of Plasmodium falciparum and Human Orotate Phosphoribosyltransferases

The survival and proliferation of Plasmodium falciparum parasites and human cancer cells require de novo pyrimidine synthesis to supply RNA and DNA precursors. Orotate phosphoribosyltransferase (OPRT) is an indispensible component in this metabolic pathway and is a target for antimalarials and antitumor drugs. P. falciparum (Pf) and Homo sapiens (Hs) OPRTs are characterized by highly dissociative transition states with ribocation character. On the basis of the geometrical and electrostatic features of the PfOPRT and HsOPRT transition states, analogues were designed, synthesized, and tested as inhibitors. Iminoribitol mimics of the ribocation transition state in linkage to pyrimidine mimics using methylene or ethylene linkers gave dissociation constants (K_d) as low as 80 nm. Inhibitors with pyrrolidine groups as ribocation mimics displayed slightly weaker binding affinities for OPRTs. Interestingly, p-nitrophenyl riboside 5′-phosphate bound to OPRTs with K_d values near 40 nm. Analogues designed with a C5-pyrimidine carbon–carbon bond to ribocation mimics gave K_d values in the range of 80–500 nm. Acyclic inhibitors with achiral serinol groups as the ribocation mimics also displayed nanomolar inhibition against OPRTs. In comparison with the nucleoside derivatives, inhibition constants of their corresponding 5′-phosphorylated transition state analogues are largely unchanged, an unusual property for a nucleotide-binding site. In silico docking of the best inhibitor into the HsOPRT active site supported an extensive hydrogen bond network associated with the tight binding affinity. These OPRT transition state analogues identify crucial components of potent inhibitors targeting OPRT enzymes. Despite their tight binding to the targets, the inhibitors did not kill cultured P. falciparum.     

A prenylation inhibitor prevents production of infectious hepatitis delta virus particles

Hepatitis delta virus (HDV) causes both acute and chronic liver disease throughout the world. Effective medical therapy is lacking. Previous work has shown that the assembly of HDV virus-like particles (VLPs) could be abolished by BZA-5B, a compound with farnesyltransferase inhibitory activity. Here we show that FTI-277, another farnesyltransferase inhibitor, prevented the production of complete, infectious HDV virions of two different genotypes. Thus, in spite of the added complexity and assembly determinants of infectious HDV virions compared to VLPs, the former are also sensitive to pharmacological prenylation inhibition. Moreover, production of HDV genotype III virions, which is associated with particularly severe clinical disease, was as sensitive to prenylation inhibition as was that of HDV genotype I virions. Farnesyltransferase inhibitors thus represent an attractive potential class of novel antiviral agents for use against HDV, including the genotypes associated with most severe disease.     

Resveratrol prevents hepatic steatosis induced by hepatitis C virus core protein

Hepatitis C virus (HCV) core protein plays an important role in the development of hepatic steatosis in patients with chronic HCV infection. Treatment of C57BL/6 mice infected with HCV core recombinant adenoviruses with resveratrol significantly decreased hepatic triacylglycerols (TAG) while the serum TAG level was unaffected. RT-PCR and Western blotting showed that HCV core protein attenuated the expression of Sirt1 and PPAR-α, which would be reversed by resveratrol. This was also confirmed in primary mouse hepatic cells infected with HCV core protein expressing adenovirus. Thus, resveratrol may prevent against hepatic steatosis by blocking the inhibited expression of Sirt1 and PPAR-α induced by HCV core protein.     

Orotate (orotic acid): An essential and versatile molecule

ABSTRACT

Orotate (OA) is well-known as a precursor in biosynthesis of pyrimidines; in mammals it is released from the mitochondrial dihydroorotate dehydrogenase (DHODH) for conversion to UMP by the cytoplasmic UMP synthase enzyme. OA is also a normal part of the diet, being found in milk and dairy products, and it is converted to uridine for use in the pyrimidine salvage pathway predominantly in liver, kidney and erythrocytes. Early research into nutrition identified orotate as “vitamin B13,” and its use as a complex with organic cations or metal ions was promulgated in body-building, and in assisting therapies of metabolic syndromes. It has recently been established that the amelioration of gout by dairy products arises from the competition of orotate and urate at the hURAT1 transporter. The orotic aciduria that arises in children with defective UMP synthase can be rescued by oral uridine therapy, since UMP is the end-product and also a feedback inhibitor of the de novo pathway. In contrast, Miller (dysmorphology) syndrome is connected with defects in DHODH, and hence in the supply of OA, and cannot be helped by uridine. Other models of dysmorphisms are connected with enzymes early in the pyrimidine de novo pathway. We conclude that the OA molecule is itself required for the regulation of genes that are important in the development of cells, tissues and organisms.     

Intensification of galactosamine hepatotoxicity by polyriboinosinic acid-polyribocytidylic acid

The effects of polyriboinosinic acid-polyribocytidylic acid (poly IC) on galactosamine-induced liver cell injury in rats were studied. Treatment of rats with D-galactosamine-HC1 (400 mg/kg) increased their serum glutamate-oxaloacetate transaminase (E.C.2.6.1.1, GOT) activities, indicating that hepatocyte injury was induced by galactosamine. Rapid and intense elevations of serum GOT activities were observed when galactosamine (200 mg/kg) was administered simultaneously with poly IC (10 mg/kg) but not with poly I or poly C. Acute increase in serum GOT activities caused by the simultaneous administration of poly IC and galactosamine was prevented by the simultaneous administration of uridine (1 g/kg), which is known to inhibit the early biochemical alterations caused by the amino sugar in the hepatocyte. These findings suggest that poly IC intensifies the hepatotoxic effect of galactosamine in rats. This poly IC-induced sensitization was inhibited by pretreatment with poly IC (10 mg/kg) itself, when injected 24 hr before the administration of the hepatotoxin together with poly IC.     

Binding of N-acetylgalactosamine-specific lectins to spin-labeled galactosamine derivatives

Legume seed lectins specific for N-acetyl-alpha-D-galactosaminyl end groups from Amphicarpaea bracteata, lima bean, Griffonia simplicifolia, Dolichos biflorus, and soybean were compared with respect to binding of several spin-labeled derivatives of D-galactosamine by electron spin resonance and precipitin inhibition analysis. Spin-label II [methyl 2-[[(2,2,5,5-tetramethyl-1-oxopyrrolidin-3-yl) carbonyl]amino]-2-deoxy-alpha-D-galactopyranoside], spin-label III [1-(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)-3-(2,2,6, 6-tetramethyl-1-oxypiperidin-4-yl)-2-thiourea], and spin-label IV [1-[4-[[(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)amino]carbonyl]phenyl]-3-(2, 2,6-tetramethyl-1-oxypiperidin-4-yl)-2-thiourea] contain 2-N-(oxypiperidinyl) or 2-N-(oxypyrrolidinyl) substituents varying in length and polarity of the linker arm between the glycoside and nitroxide ring. Spin-labels II and III were found to bind very weakly to all the lectins tested (Kd greater than or equal to 1.0 mM). Spin-label IV, containing a planar, nonpolar 2-N-phenyl group, was bound very strongly (Kd = 0.1-0.4 mM) and was moderately immobilized (2T parallel = 48-56 G) by all lectins except that from D. biflorus. Notably, the affinity of spin-label IV to lima bean lectin was 18-fold greater than that for methyl N-acetyl-alpha-galactosaminide. These results suggest that when the bulky oxypiperidinyl moiety lies in a position close to the sugar ring, it interferes with binding; in the cases where a phenyl group spacer exists, the aromatic ring in some cases actually enhances binding.(ABSTRACT TRUNCATED AT 250 WORDS)