Functional characterization of BC039389-GATM and KLK4-KRSP1 chimeric read-through transcripts which are up-regulated in renal cell cancer.

BACKGROUND: Chimeric read-through RNAs are transcripts originating from two directly adjacent genes (<10 kb) on the same DNA strand. Although they are found in next-generation whole transcriptome sequencing (RNA-Seq) data on a regular basis, investigating them further has usually been refrained from. Therefore, their expression patterns or functions in general, and in oncogenesis in particular, are poorly understood. RESULTS: We used paired-end RNA-Seq and a specifically designed computational data analysis pipeline (FusionSeq) to nominate read-through events in a small discovery set of renal cell carcinomas (RCC) and confirmed them in a larger validation cohort. 324 read-through events were called overall; 22/27 (81%) selected nominees passed validation with conventional PCR and were sequenced at the junction region. We frequently identified various isoforms of a given read-through event. 2/22 read-throughs were up-regulated: BC039389-GATM was higher expressed in RCC compared to benign adjacent kidney; KLK4-KRSP1 was expressed in 46/169 (27%) RCCs, but rarely in normal tissue. KLK4-KRSP1 expression was associated with worse clinical outcome in the patient cohort. In cell lines, both read-throughs influenced molecular mechanisms (i.e. target gene expression or migration/invasion) in a way that counteracted the effect of the respective parent transcript GATM or KLK4. CONCLUSIONS: Our data suggests that the up-regulation of read-through RNA chimeras in tumors is not random but causes regulatory effects on cellular mechanisms and may impact patient survival.

Gyrate atrophy of the choroid and retina with hyperornithinemia. Deficient formation of guanidinoacetic acid from arginine.

Patients with gyrate atrophy of the choroid and retina have 10- to 20-fold increased ornithine concentrations in body fluids and significantly reduced activity of ornithine aminotransferase in lymphocytes and cultured fibroblasts. We administered intravenous arginine to six patients and six controls to study in vivo inhibition by high ornithine concentrations of arginine-glycine transamidinase, the rate-limiting enzyme in creatine synthesis. Serum arginine concentrations curves after administration were similar for the two groups. The increment in serum ornithine was more than three times as great in patients as in controls. The mean half-times in plasma ornithine were 360 and 97 min, respectively. In the patients, the metabolic clearance of ornithine from the extracellular fluid was significantly delayed. Urinary guanidinoacetate excretion rose markedly in all controls, the excretion rate being higher in females. The patients always excreted less than the controls, the differences within the sexes being highly significant. Differences in creatine excretion after administration were less marked. We conclude that in gyrate atrophy patients, formation of guanidinoacetate, creatine, and possibly phosphocreatine is inhibited at the transaminidation step by the high concentrations of ornithine. Deficiency of the high-energy phosphates may underlie the pathogenesis of the eye and muscle atrophies.

[Gas chromatography for diagnosis and prognosis of destructive pancreatitis].

One of modern and highly effective methods of diagnostics and differential diagnostics of infected pancreonecrosis is chromatographic technique, which allows for identification of anaerobic non-clostridial infection in the foci of pancreatic destruction by the presence of volatile fatty acids, the specific end products of anaerobic bacterial metabolism. Gas chromatography (GC) - mass spectrometry (MS) also make it possible to detect in peripheral blood of patients with pancreatitis certain metabolites (di-, polyamines, and aromatic amines) that are markers of tissue (protein structure) disintegration and the degree of pancreonecrosis, which is a valuable indicator of the degree of pancreonecrosis. The presence, according to GC - MS analysis, of natural inhibitors of transamidinase (compounds of thiourea and its metabolites, the group of mercaptopurines and mercapto-derivates of imidazole) in peripheral blood at the maximum level--0.71 to 0.78 mmol/l on days 7 to 10 upon the onset of the disease--is a prognostically favorable criterion. At the same time, the presence of the maximum level of anaerobic non-clostridial infection metabolite in peripheral blood 1.12 to 1.31 mmol/l on days 7 to 10 upon the onset of the disease--is prognostically infavorable. These chromatographic criteria in combination with clinical manifestations can be considered indications to surgical treatment of infected pancreonecrosis, and the prognosis of the disease can be based on them.

Inhibition of arginine-glycine amidinotransferase by ornithine. A possible mechanism for the muscular and chorioretinal atrophies in gyrate atrophy of the choroid and retina with hyperornithinemia.

The inhibitory effect of ornithine on L-arginine:glycine amidinotransferase (EC 2.1.4.1) was studied in crude rat kidney homogenates. The enzyme activity was linear with time up to 45 min and with protein up to 200 microgram. The apparent Km and V of amidinotransferase were 9.21 mM and 1.53 mu mol/g protein per min, respectively. The enzyme was competitively inhibited by ornithine, with a Ki of 0.253 mM. Kidney arginase was inhibited only slightly and non-competitively. The inhibition of amidinotransferase by ornithine may thus be important in creatine biosynthesis. In gyrate atrophy of the choroid and retina with hyperornithinemia, a human autosomal recessive disease caused by decreased ornithine aminotransferase activity, plasma ornithine concentrations are elevated 10-20-fold (0.65-1.35 mM during fasting). In consequence endogenous creatine production probably is severely decreased because of inhibition of the rate-limiting transamidination step by ornithine. The deficiency of creatine and further of readily available energy in the form of phosphocreatine is suggested to be involved in the pathogenesis of the choroidal, retinal and type II muscle fiver atrophies in gyrate atrophy.

Creatine biosynthesis during embryonic development. False feedback suppression of liver amidinotransferase by N-acetimidoylsarcosine and 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine).

The level of arginine:glycine amidinotransferase in liver of the developing chick embryo is partially suppressed following injection of arginine into the yolk, and the level can be completely suppressed following injection of guanidinoacetate or creatine (Walker, J.B. (1963), Proc. Soc. Exp. Biol. Med. 112, 245; Walker, J.B., and Wang, S.-H. (1964), Biochim, Biophys. Acta 81, 435). In this investigation structural requirements for the physiological suppressor were examined by testing certain analogues of creatine and its biosynthetic precursors for their ability to suppress liver amidinotransferase levels in developing chick embryos and growing chicks. The creatine analogues, N-acetimidoylsarcosine and 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine), were found to suppress liver amidinotransferase levels of both developing embryos and growing chicks. Compounds ineffective as suppressors included: the arginine analogue, N5-acetimidoylornithine; the guanidinoacetate analogue, N-acetimidoylglycine; and the creatine analogue, 1-carboxymethyl-2-iminohexahydropyrimidine. Our findings suggest that (i) arginine and guanidinoacetate must be converted to creatine before serving as a suppressor, and (ii) creatine, not phosphocreatine, is most closely related to the physiological suppressor of amidinotransferase.

Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions.

A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine--Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis--Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine--hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated.