UGA is read as tryptophan in Mycoplasma capricolum.

UGA is a nonsense or termination (opal) codon throughout prokaryotes and eukaryotes. However, mitochondria use not only UGG but also UGA as a tryptophan codon. Here, we show that UGA also codes for tryptophan in Mycoplasma capricolum, a wall-less bacterium having a genome only 20-25% the size of the Escherichia coli genome. This conclusion is based on the following evidence. First, the nucleotide sequence of the S3 and L16 ribosomal protein genes from M. capricolum includes UGA codons in the reading frames; they appear at positions corresponding to tryptophan in E. coli S3 and L16. Second, a tRNATrp gene and its product tRNA found in M. capricolum have the anticodon sequence 5' U-C-A 3', which can form a complementary base-pairing interaction with UGA.     

CGG: an unassigned or nonsense codon in Mycoplasma capricolum.

CGG is an arginine codon in the universal genetic code. We previously reported that in Mycoplasma capricolum, a relative of Gram-positive eubacteria, codon CGG did not appear in coding frames, including termination sites, and tRNA(ArgCCG) pairing with codon CGG, was not detected. These facts suggest that CGG is a nonsense (unassigned and untranslatable) codon--i.e., not assigned to arginine or to any other amino acid. We have investigated whether CGG is really an unassigned codon by using a cell-free translation system prepared from M. capricolum. Translation of synthetic mRNA containing in-frame CGG codons does not result in "read-through" to codons beyond the CGG codons--i.e., translation ceases just before CGG. Sucrose-gradient centrifugation profiles of the reaction mixture have shown that the bulk of peptide that has been synthesized is attached to 70S ribosomes and is released upon further incubation with puromycin. The result suggests that the peptide is in the P site of ribosome in the form of peptidyl-tRNA, leaving the A site empty. When in-frame CGG codons are replaced by UAA codons in mRNA, no read-through occurs beyond UAA, just as in the case of CGG. However, the synthesized peptide is released from 70S ribosomes, presumably by release factor 1. These data suggest strongly that CGG is an unassigned codon and differs from UAA in that CGG is not used for termination.     

Coordinate regulation of unsaturated phospholipid, RNA, and protein synthesis in Mycoplasma capricolum by cholesterol.

The effect of cholesterol, epicoprostanol, and phosphatidylcholine on phospholipid, RNA, and protein synthesis was investigated in the sterol auxotroph Mycoplasma capricolum. Cells growing poorly on lanosterol were stimulated to grow more rapidly by supplementing the medium with either 2 micrograms of cholesterol or 2.2 micrograms of egg phosphatidylcholine per ml. In such cells cholesterol caused a sequential stimulation of phospholipid, RNA, and protein synthesis. Enhanced oleate incorporation into phospholipid occurred early; the rates of RNA and protein synthesis increased later. In cells supplemented with phosphatidylcholine only RNA and protein syntheses were enhanced. The addition of 2 micrograms of epicoprostanol per ml to cells growing on lanosterol promptly inhibited the rate of unsaturated phospholipid synthesis and subsequently the rate of growth. Inhibition of both processes was relieved by supplying 2 micrograms of cholesterol or 2.2 micrograms of phosphatidylcholine per ml along with the inhibitory sterol. The results suggest that cholesterol in small amounts exerts a positive regulatory effect and epicoprostanol exerts a negative one on unsaturated phospholipid synthesis and, in turn, that RNA and protein synthesis are coordinately controlled with phospholipid synthesis. The previously reported phenomenon of sterol synergism and the postulated novel role of sterols in membranes.     

Occurrence of unmodified adenine and uracil at the first position of anticodon in threonine tRNAs in Mycoplasma capricolum.

Codon usage pattern in the threonine four-codon (ACN) box in Mycoplasma capricolum is strongly biased towards adenine and uracil for the third base of codons. Codons ending in uracil or adenine, especially ACU, predominate over ACC and ACG. This bacterium contains two isoacceptor threonine tRNAs having anticodon sequences AGU and UGU, both with unmodified first nucleotides. It would thus appear that ACN codons are translated in an unusual way; tRNA(Thr)(AGU) would translate the most abundantly used codon ACU exclusively, because adenine at the first anticodon position can, according to the wobble rule, pair only with uracil of the third codon position. The tRNA(Thr)(UGU) would mainly be responsible for translation of three other codons, ACA, ACG, and ACC. Anticodon UGU would also be used for reading codon ACU as a redundancy of tRNA(Thr)-(AGU), as deduced from the mitochondrial code where unmodified uracil at the first anticodon position can pair with adenine, cytosine, guanine, and uracil by four-way wobble. The tRNA(Thr)(AGU) has much higher sequence homology to tRNA(Thr)(UGU) from M. capricolum (88%), Bacillus subtilis (77%) and Escherichia coli (86%) than to tRNA(Thr)(GGU) from B. subtilis (66%) and E. coli (63%), suggesting that tRNA(Thr)-(AGU) has been derived from tRNA(Thr)(UGU), but not from tRNA(Thr)(GGU).     

Sterol synergism in yeast.

Sterol synergism as previously observed [Dahl, C.E., Dahl, J.S. & Bloch, K. (1980) Biochemistry 19, 1462-1467] and defined as a greater-than-additive growth response to pairs of sterols by Mycoplasma capricolum [Dahl, J.S., Dahl, C.E. & Bloch, K. (1981) J. Biol. Chem. 256, 87-91] is now demonstrated in the yeast mutant GL7, which is auxotrophic for sterol and unsaturated fatty acid. Mutant cells growing poorly when provided with cholesterol and oleic acid respond to ergosterol supplements (ergosterol-to-cholesterol ratio, 1:3) by a pronounced increase in growth rates and cell yields. Stigmasterol also elicits a significant synergistic effect, and 7-dehydrocholesterol, a smaller one. Evidence for a metabolic role of ergosterol in yeast membranes is presented. Cells raised on a 1:3 mixture of ergosterol to cholesterol up to midlogarithmic phase subsequently incorporate [1-14C]oleic acid at significantly faster rates into phospholipids than do cells grown on cholesterol alone.     

Mycoplasma arthritidis mitogen. V beta engaged in mice, rats, and humans, and requirement of HLA-DR alpha for presentation.

OBJECTIVE. Mycoplasma arthritidis mitogen (MAM) is mitogenic for mouse, rat, and human T cells, and behaves as a superantigen in mice through its capacity to bind to the alpha chain of I-E molecules and engage entire sets of T cells expressing specific V beta. Here, we have attempted to fully characterize the V beta-engaging activities of MAM in mice, and define similar activities in rats and humans. METHODS. Multiprobe RNase-protection assays and mice transgenic for human DR alpha, DR beta, and DR alpha beta were utilized for this purpose. RESULTS. MAM-reactive V beta in the mouse included not only the previously reported V beta 6, V beta 8.1, V beta 8.2, and V beta 8.3, but also V beta 5.1. In the rat, engagement of V beta 5.1, V beta 6, V beta 8.1, and V beta 8.2, but not V beta 8.3, was documented, whereas in humans, the engaged V beta included primarily V beta 19.1 (alternatively termed V beta 17.1) and, to a lesser extent, V beta 3.1, V beta 11.1, V beta 12.1, and V beta 13.1. In DR transgenic E alpha- E beta- mice, presentation of MAM and engagement of specific V beta was effected by DR alpha. CONCLUSIONS. Homologous V beta are engaged by MAM in mice, rats, and humans, presumably through a binding site similar to that proposed previously for other superantigens. MAM presentation primarily via the nonpolymorphic DR alpha makes it unlikely that there is involvement of such a superantigen in the pathogenesis of autoimmune diseases known to be associated with certain DR haplotypes. The possibility cannot be excluded, however, that superantigen-activated T cells may lead to disease by cross-reactions with self-antigens presented by particular DR haplotypes.     

Sterol balance in a patient with abetalipoproteinaemia.

Total-body cholesterol synthesis was measured in a woman with abetalipoproteinaemia and in a normal woman of similar age. The rate of synthesis of cholesterol was 15.4 +/- 4.1 mg/kg/day in the patient and 14.3 +/- 2.6 mg/kg/day in the control subject, indicating that cholesterol synthesis in the whole body is not increased in the complete absence of plasma low density lipoprotein.     

Life-threatening Mycoplasma hominis mediastinitis.

Mycoplasma hominis infections are easily missed because conventional methods for bacterial detection may fail. Here, 8 cases of septic mediastinitis due to M. hominis are reported and reviewed in the context of previously reported cases of mediastinitis, sternum wound infection, pleuritis, or pericarditis caused by M. hominis. All 8 patients had a predisposing initial condition related to poor cardiorespiratory function, aspiration, or complications related to coronary artery surgery or other thoracic surgeries. Mediastinitis was associated with purulent pleural effusion and acute septic symptoms requiring inotropic medication and ventilatory support. Later, the patients had a tendency for indolent chronic courses with pleuritis, pericarditis, or open sternal wounds that lasted for several months. M. hominis infections may also present as mild sternum wound infection or as chronic local pericarditis or pleuritis without septic mediastinitis. Treatment includes surgical drainage and debridement. Antibiotics effective against M. hominis should be considered when treating mediastinitis of unknown etiology.     

Sterol phylogenesis and algal evolution

The stereochemistry of several sterol precursors and end products synthesized by two fungal-like micro-organisms Prototheca wickerhamii (I) and Dictyostelium discoideum (II) have been determined by chromatographic (TLC, GLC, and HPLC) and spectral (UV, MS, and 1H NMR) methods. From I and II the following sterols were isolated from the cells: cycloartenol, cyclolaudenol, 24(28)-methylenecycloartanol, ergosterol, protothecasterol, 4alpha-methylergostanol, 4alpha-methylclionastanol, clionastanol, 24beta-ethylcholesta-8,22-enol, and dictyosterol. In addition, the mechanism of C-24 methylation was investigated in both organisms by feeding to I [2-3H]lanosterol, [2-3H]cycloartenol, [24-3H]lanosterol, and [methyl-2H3]methionine and by feeding to II [methyl-2H3]methionine. The results demonstrate that the 24beta configuration is formed by different alkylation routes in I and II. The Delta25(27) route operates in I while the Delta24(28) route operates in II. Based on what is known in the literature regarding sterol distribution and phylogenesis together with our findings that the stereochemical outcome of squalene oxide cyclization leads to the production of cycloartenol rather than lanosterol (characteristic of the fungal genealogy) and the chirality of the C-24 alkyl group is similar in the two nonphotosynthetic microbes (beta oriented), we conclude that Prototheca is an apoplastic Chlorella (i.e., an alga) and that Dictyostelium as well as the other soil amoebae that synthesize cycloartenol evolved from algal rather than fungal ancestors.     

Inhibition of Sterol Biosynthesis by Bacitracin

Bacitracin is an inhibitor of the biosynthesis of squalene and sterols from mevalonic acid, C(5)-isopentenyl pyrophosphate, or C(15)-farnesyl pyrophosphate catalyzed by preparations from rat liver. The antibiotic is active at extremely low ratios of antibiotic to substrate. The mechanism of inhibition appears to be the formation of a complex between bacitracin, divalent cation, and C(15)-farnesyl pyrophosphate and other isoprenyl pyrophosphates. It is similar to the formation of the complex with C(55)-isopropenyl pyrophosphate in microbial systems. The toxicity of bacitracin for animal cells could be due in part to the formation of these complexes.     

Mycoplasma antibodies in rheumatoid arthritis.

Sera of 100 patients under examination at the Outpatient Department of the Rheumatism Foundation Hospital were studied by the indirect hemagglutination technique, using both mycoplasma reference strains, and isolates from RA and SLE as antigens. The series consisted of five groups: I, definite RA (49 patients); II, probable RA (11); III, possible RA or nonspecific inflammatory arthritis (34); IV, osteoarthrosis (2); V, Reiter's disease (4). Mycoplasma antibodies in titres of 16 or higher were encountered in groups I-IV in 26, 8, 19, and one case respectively. Twenty-one out of 106 blood donors had antibodies against an isolate from RA and/or M. arthritidis strain PG 6. The titres found were 16 or 32, except in two cases, 128. In the definite RA group, 21 out of 26 patients possessing mycoplasma antibodies, showed titres of 16 or higher against isolates from RA and/or SLE, 12 against M. arthritidis strain PG 6 and/or Campo, 8 against M. fermentans, and 6 against a T-strain from NGU. Antibodies against M. arthritidis strain Campo were found more often than against strain PG 6. The longer the duration of the arthritic symptoms was, the more frequent seemed also to be the occurrence of mycoplasma antibodies.     

Ocular manifestations of Mycoplasma pneumoniae infection.

Ocular manifestations of Mycoplasma pneumoniae infection, other than conjunctivitis, are uncommon. Optic disk swelling, optic nerve atrophy, retinal exudates and hemorrhages, and cranial nerve palsies have been infrequently reported. We describe a 15-year-old patient who developed bilateral optic disk edema and iritis during an acute infection with M. pneumoniae and review the world literature on findings associated with ocular manifestations of infection with this pathogen. Although our patient experienced complete resolution of iritis and optic disk edema after 6 weeks, several patients described in the literature have experienced permanent sequelae as a result of optic neuropathy.     

Sterol Metabolism in the Rat: Effect of Alcohol on Sterol Metabolism in Two Strains of Rats

Sterol metabolism studies usinag a combination of iso-topic and chromatographic procedures were carried out in two strains of rats fed 5% ethanol (36% of calories) in the diet.
Feeding ethanol to the Fisher rat over 17 days produced no significant changes in body weight. Cholesterol levels in various tissues were elevated in the ethanol-fed group: plasma cholesterol, +61%; liver cholesterol, +47%; and bile cholesterol, +57%. The alcohol-fed Fisher rat showed several changes in sterol metabolism over controls: fecal acidic steroid output, + 13%; fecal neutral sterol output, +51%; endogenous neutral sterol output, +107%; cholesterol turnover, + 54%; and cholesterol balance, +18%.
Ethanol feeding to the Spraque Dawley rat showed similar differences between ethanol-fed vs. control rats. Cholesterol levels were significantly elevated in plasma (+35%) and in the liver (+81%). Sterol metabolism data showed the following differences (alcohol vs. control): fecal acidic steroid output, +9%; fecal neutral sterol output, +17%; endogenous neutral sterol output, +72%; cholesterol turnover, +33%; and cholesterol balance, +13%.
The Fisher rat maintained almost constant weight throughout the experimental period and is a preferable strain for sterol balance studies using liquid diets. A major finding of these experiments was the increased concentration of cholesterol in liver, plasma, and bile in both strains of rats. The sterol balance measurements indicated that this tissue accumulation of cholesterol was due to enhanced cholesterol synthesis as well as inhibition of bile acid synthesis.     

Sterol control of the phosphatidylethanolamine-phosphatidylcholine conversion in the yeast mutant GL7.

The relatively slow growth rate of the yeast mutant GL7, a sterol auxotroph, on medium containing cholesterol is markedly accelerated by supplementation with small amounts of ergosterol. Under these conditions (sterol synergism) cellular phospholipid synthesis is enhanced. We now find that one of the ergosterol-stimulated processes is the methylation of phosphatidylethanolamine to phosphatidylcholine. This is shown by comparing methyltransferase activities of membrane preparations derived from cells grown on either ergosterol, cholesterol, or the synergistic sterol pair. Incorporation of 32P from [gamma-32P]ATP into the yeast membranes is rapid and greater when ergosterol-grown cells rather than cholesterol-grown cells are the source of membranes.