Eukaryotic tRNAs(Pro): primary structure of the anticodon loop; presence of 5-carbamoylmethyluridine or inosine as the first nucleoside of the anticodon.

The modified nucleoside U*, located in the first position of the anticodon of yeast, chicken liver and bovine liver tRNA(Pro) (anticodon U*GG), has been determined by means of TLC, HPLC, ultraviolet spectrum and gas chromatography-mass spectrometry. The structure was established as 5-carbamoylmethyluridine (ncm5U). In addition, we report on the primary structures of the above-mentioned tRNAs as well as those which have the IGG anticodon. In yeast, the two tRNA(Pro) (anticodons U*GG and IGG) differ by eight nucleotides, whereas in chicken and in bovine liver, both anticodons are carried by the same 'body tRNA' with one posttranscriptional exception at position 32, where pseudouridine is associated with ncm5U (position 34) in tRNA(Pro) (U*GG) and 2'-O-methylpseudouridine is associated with inosine (position 34) in tRNA(Pro) (IGG).

The DIM1 gene responsible for the conserved m6(2)Am6(2)A dimethylation in the 3'-terminal loop of 18 S rRNA is essential in yeast.

Biogenesis of cytoplasmic ribosomes universally involves methylation of ribosomal RNA. Little genetic evidence is available about the functional role(s) of this conserved posttranscriptional modification. The only known methylase gene involved in rRNA maturation is ksgA in Escherichia coli, which directs dimethylation of two adjacent adenosines (m6(2)A1518m6(2)A1519) in the loop of a conserved hairpin near the 3'-end of 16 S rRNA. This tandem methylation is the only rRNA modification common to pro and eukaryotes. Disruption of ksgA confers resistance to the aminoglycoside antibiotic kasugamycin without significantly impairing viability. Here we report the cloning of the DIM1 gene encoding the homolog 18 S rRNA dimethylase in Saccharomyces cerevisiae. The yeast enzyme is evolutionary related to the ksgA protein. It carries a distinctive lysine-rich-N-terminal extension with a potential protein kinase C phosphorylation site. Like ksgA, DIM1 belongs to the erm family of prokaryotic 23 S rRNA dimethylases responsible for erythromycin resistance. Surprisingly, disruption of DIM1 turns out to be lethal in yeast.

Evolutionary changes in CpG and methylation levels in the genome of vertebrates.

We have analysed the levels of 5-methylcytosine (5mC) in DNAs from 42 vertebrates, and compiled, including data from literature, a table of genomic 5mC and GC levels (as well as the available c-values, i.e., the haploid genome sizes) of 87 species from all vertebrate classes. An analysis of the data indicates that (i) two positive correlations hold between the 5mC and GC levels of the genomes of fishes/amphibians and mammals/birds, respectively; (ii) the genomes of fishes and amphibians are, on average, about twice as methylated as those of mammals, birds and reptiles, this difference being unrelated to the amounts of repetitive DNA sequences; (iii) the 5mC and CpG observed/expected values show no overlap between the two groups of vertebrates and suggest the existence of two equilibria. The transition separating the two equilibria appears to have taken place at the time of appearance of reptiles. Its possible cause(s) and its implications are discussed.

Methylation patterns in the isochores of vertebrate genomes.

5-Methylcytosine (5mC) levels were determined in compositional DNA fractions corresponding to different isochore families from the genomes of Xenopus, chicken, mouse and human, four vertebrates which show different isochore patterns. The results obtained indicate that: (i) positive correlations exist between the 5mC levels and the GC levels of isochores within any given genome; and (ii) DNA from Xenopus isochore families is twice as methylated as DNA from the isochores having the same GC levels from mouse, human and chicken. Moreover, the positive correlations holding between CpG levels and the GC3 levels of coding sequences of warm-blooded vertebrates were shown to comprise two regions with a border at approx. 75% GC3. The correlation corresponding to the higher region (which comprises only very rare high GC3 values in the case of Xenopus) has a higher slope than that corresponding to the lower GC3 values, a phenomenon due in all likelihood, to the increasing contribution of CpG islands. Finally, the observed/expected CpG ratio is higher in Xenopus than in warm-blooded vertebrates.

The tRNA N2,N2-dimethylguanosine-26 methyltransferase encoded by gene trm1 increases efficiency of suppression of an ochre codon in Schizosaccharomyces pombe.

In the majority of eukaryotic tRNAs, the guanosine at position 26 is modified by a dimethyl group, but so far a function of this modification has not been detected. We isolated the Schizosaccharomyces pombe gene, trm1, encoding the tRNA N2, N2-dimethylguanosine-26 methyltransferase. Strains having the gene deleted completely lack N2,N2-dimethylguanosine. In strains carrying the weak ochre tRNA suppressor sup3-i, deletion of trm1 abolishes suppression indicating that the trm1 deletion acts as an antisuppressor mutation. The result suggests that in vivo N2, N2-dimethylguanosine-26 increases the capacity of the sup3-i serine tRNA to translate the UAA (ochre) codon.

Presence and coding properties of 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um) in the wobble position of the anticodon of tRNA(Leu) (U*AA) from brewer's yeast.

The unknown modified nucleoside U* has been isolated by enzymatic and HPLC protocols from tRNA(Leu) (U*AA) recently discovered in brewer's yeast. The pure U* nucleoside has been characterized by electron impact mass spectroscopy, and comparison of its chromatographic and UV-absorption properties with those of appropriate synthetic compounds. The structure of U* was established as 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um). The yeast tRNA(Leu) (U*AA) is the only tRNA so far sequenced which has been shown to contain ncm5Um. The location of such a modified uridine at the first position of the anticodon restricts the decoding property to A of the leucine UUA codon.

Sequence of a new tRNA(Leu)(U*AA) from brewer's yeast.

The nucleotide sequence of a new tRNA(Leu)(anticodon U*AA) from Saccharomyces cerevisiae which could recognize exclusively the UUA codon has been determined. Its primary structure is: pGGAGGGUUGm2GCac4CGAGDGmGDCDAAGGCm2(2)GGCAGACmUU*AAm1GA++ + psi CUGUUGGACGGUUGUCCGm5CGCGAGT psi CGm1A(orA)ACCUCGCAUCCUUCACCA. This tRNA has a large extraloop and contains 15 modified nucleotides. So far it is the third isoacceptor tRNA for leucine in yeast. It has 61% homology with tRNA(Leu)(anticodon m5CAA) and 63% homology with tRNA(Leu)(anticodon UAG), the two other known yeast tRNAs(Leu).

Combined nutritional support and continuous extracorporeal removal therapy in the severe acute phase of maple syrup urine disease.

OBJECTIVE: The authors assessed the efficiency, tolerance and outcome of neonates and children with maple syrup urine disease (MSUD) in acute decompensation managed by endogenous and extracorporeal removal of accumulated MSUD metabolites. DESIGN: Single center cohort study. SETTING: Pediatric and neonatal intensive care unit in a tertiary care hospital. PATIENTS: Between January, 1991, and June, 1999, six neonates and six children in acute decompensation of MSUD were included in the study. Each of them had two of the three following criteria: comatose state, gastrointestinal intolerance, leucine plasma levels over 1700 micromol/l. INTERVENTIONS: Patients were treated by combined nutrition manipulation and continuous venovenous extracorporeal removal therapies (CECRT) including hemofiltration, hemodialysis or hemodiafiltration. A clinical and biological evaluation was performed before, during and following the treatment. RESULTS: Eleven out of the 12 patients survived. One child had two acute episodes at 6.5 and 9 years old. Eight patients recovered a normal cerebral performance category score. In all cases, plasma leucine level decreased according to a logarithmic mode within 11-24 h hemodiafiltration combined with nutritional support whereas, with nutrition alone after stopping CECRT, the decrease in leucine plasma levels was slower, following a linear mode. Eight patients were supplemented with valine and isoleucine for mean plasma values of 177+/-92 and 68+/-66, respectively. CONCLUSION: In severe acute decompensation of MSUD, CECRT combined with nutritional support limit central nervous system damage, by dramatically decreasing branched chain amino and keto acid levels.

Are branched chain amino-acids beneficial for nutrition of severe surgical patients with sepsis?

UNLABELLED: This study investigated in severe sepsis the metabolic effects of two amino acid diets, including the same protein (0.3 gN/kg/day) and caloric intake (2g/kg/day glucose) but with different amino acid composition, 24% branched chain amino-acids (B.C.A.A.) for diet A (8 cases), 41% B.C.A.A. for diet B (11 cases). The two groups were obtained by randomisation at the operative day (D0) and were observed six days (D1-D6). During study, total nitrogen using a Kjeldhal method was measured. At D0 and D6, albumin, total hemolytic complement, transferrin, cholesterol, ammonaemia, insulin and amino acid pattern were studied in blood and skin tests were performed. The data were analysed by Mann-Whitney test. RESULTS: 1) The cumulative nitrogen balances of the six days are not significantly different, but at day 6 in diet B, patients started to be in an anabolic state (+3.7 gN) and in diet A catabolism persisted (-12 gN) (p<0.02). 2) Plasma B.C.A.A. are significantly higher and aromatic acids significantly lower in diet B. In order, Fischer ratio is also significantly different between the two regimens. All the other measured parameters are not significantly different. In this study, the B.C.A.A. enriched diet seems to be beneficial in surgical patients with severe sepsis.

[Neuroendocrine disorders observed in the post-critical phase in epileptic patients]. / Les troubles neuro-endocriniens observés en phase post-critique chez les épileptiques.

In 10 epileptic patients with generalized seizures, plasma levels of ACTH, prolactin, FSH, LH, TSH, were measured first within 60 minutes after the crisis, then 3 to 5 days later without changes in antiepileptic drugs. Within 1 hour after the seizure, a significant rise of ACTH and prolactin 3 - 4 folds the levels observed in the 2nd measure was present. This was compared to measures made within 1 hour after a syncope in which case it was not present. The post-critic rise of ACTH and prolactin would appear to be a characteristic of generalized epileptic seizures.

[Removal of amino acids by continuous hemofiltration and hemodiafiltration]. / Epuration des acides aminés par hémofiltration et hémodiafiltration continues.

BACKGROUND: Continuous hemodiafiltration is potentially more efficient than hemofiltration for removing low molecular metabolites. CASE REPORT: A newborn (2100 g) was admitted suffering from acute distress with statuts epilepticus, liver failure with generalized hyperaminoacidemia, hypoglycemia and oligo-anuria. Peritoneal dialysis, performed on the 17th day of life, was ineffective and the patient was treated by continuous hemofiltration alternating with hemodiafiltration. Clearance of amino-acids was studied for 15 minutes under each technique. The amino-acid concentrations were measured in the infused fluid and in the ultrafiltrate. The baby died on the 20th day despite this treatment. RESULTS: Amino acid clearance by hemodiafiltration was 181 +/- 176% greater than by hemofiltration. The mean improvement for all amino acids was 148%, with extremes of +43% for citrulline and 941% for glutamic acid. CONCLUSION: Continuous hemodiafiltration is an efficient method of removing amino acids; it could be used to treat severe inborn errors of metabolism such as leucinosis.

[GABAergic control of hypophyseal gonadotropic function and prolactin. Demonstration by valproic acid]. / Le contrôle gabaergique de la fonction gonadotrope hypophysaire, et de la prolactine. Mise en évidence par l'acide valproïque.

While there is well-known evidence for dopaminergic control over gonadotrophic and Prolactin functions, the way gamma-aminobutyric acid (Gaba) acts and the routes through which it acts are not so well known. Valproic acid stimulates cerebral Gabaergic tracts. It was therefore used by the authors to study first the basal levels of LH FSH and Prolactin and the way they reacted after two months treatment with this anti-convulsant. The authors noted a decrease of LH-RH stimulated LH and a decrease of the basal levels or Prolactin in TRH stimulated Prolactin. So Gaba can be a neuro-modulator of the hypothalamo-pituitary axis, acting synergically with the dopaminergic tracts to decrease Prolactin secretion but opposing the dopaminergic tracts in the control over LH.

[Post-crisis elevation of adrenocorticotropic hormone and prolactin in epileptic children]. / Elévation post-critique de l'hormone adrénocorticotrope et de la prolactine chez les enfants épileptiques.

In six children, aged from 8 to 12 years, presenting with primary generalized epilepsy a significant rise in plasma ACTH and prolactin levels (as compared with levels measured 5 days later) was observed either during a generalized seizure demonstrated by EEG (3 cases) or within 1 hour of a generalized seizure (3 cases). The neurophysiological basis for these post-epileptic endocrine disturbances is a sudden depression of dopaminergic and GABA-ergic pathways. As a result, these endocrine changes, already well documented in adults, may be regarded as a good biological marker of the epileptic origin of a paroxysmal attack.

[Endocrine abnormalities in HIV infections]. / Anomalies endocriniennes au cours de l'infection par le VIH.

The finding of endocrine gland lesions at pathological examination in AIDS and reports of several cases of endocrine disease in patients with this syndrome have prompted us to study endocrine functions in 63 patients (51 men, 12 women) with HIV-1 infection. According to the Center for Disease Control (CDC) classification system, 13 of these patients were stage CDC II, 27 stage CDC III and 23 stage CDC IV. We explored the adrenocortical function (ACTH, immediate tetracosactrin test) and the thyroid function (free T3 and T4 levels, TRH on TSH test) in all 63 patients. The hypothalamic-pituitary-gonadal axis (testosterone levels, LHRH test) and prolactin secretion (THR test) were explored in the 51 men. The results obtained showed early peripheral testicular insufficiency at stage CDC II and early pituitary gland abnormalities with hypersecretion of ACTH and prolactin also at stage CDC II. On the other hand, adrenocortical and pituitary abnormalities were not frequently found. The physiopathology of the endocrine abnormalities observed in HIV-1-infected patients remains unclear, but one may suspect that it involves interleukin-1 since this protein factor has recently been shown to stimulate the corticotropin-releasing hormone secretion and to act directly on the glycoprotein capsule of the virus (gp 120) whose structure is similar to that of some neurohormones.