[Agranulocytosis caused by dipyrone. Case reports]. / Agranulocitosi da dipirone: contributo casistico.

Two cases of dipyrone-induced agranulocytosis are described. In the first one, the disease was due to a single administration, in the second one, to prolonged therapy. The possible pathogenic mechanism is discussed, which can be immunologic and/or toxic. The effectiveness of some of the drugs and general supportive measures applied is discussed. Health education of the patient, in conjunction with careful attention by the physician, may play an important role in the prevention of this pathological condition.

In vitro synthesis of ornithine transcarbamylase.

An in vitro system for the synthesis of ornithine transcarbamylase (OTCase) was established using iS-30 extract from E. coli MDS6-2(lambda) and DNA of a lambda transducing phage carrying argI and argF genes. This in vitro synthesis was completely dependent on the additon of DNA, and was sensitive to chloramphenicol and rifampicin. Radioisotopic analysis confirmed that the synthesized enzyme catalyzes the carbamylation of ornithine to citrulline. In the in vitro system the repression and derepression of OTCase synthesis could be observed by mixing iS-30 extracts prepared from argR+ and argR- cells. A remarkable maturation effect could be observed for the FFF enzyme, but not for the III enzyme. This system is considered to reflect the in vivo situation, and should therefore be useful for investigations on the regulation of OTCase synthesis in vivo.

Some regulation profiles of ornithine transcarbamylase synthesis in vitro.

The regulation profiles of OTCase (argF, argI) synthesis in vitro were investigated by using the in vitro system described in the accompanying paper. Addition of 2.6 mM arginine, crude repressor and partially purified repressor to the in vitro system demonstrated that lambdadargF-DNA-directed OTCase-FFF synthesis is more sensitive to the repressor than lambdapargI-DNA-directed OTCase-III synthesis. The effects of some low-molecular substances on FFF and III syntheses were investigated; guanosine 3'-diphosphate 5'-diphosphate (ppGpp) stimulated both syntheses while cAMP and guanosine 5'-tetraphosphate (Gpppp) were not effective on III synthesis and were slightly inhibitory for FFF synthesis. The substances had no effect on the maturation of the enzyme or on the activity of the enzyme, FFF or III, synthesized. We suggest that argF- and argI-genes are regulated in a slightly different fashion and that the operator-promotor regions are not completely identical for these two genes.

Interaction between mutations of ribosomes and RNA polymerase: a pair of strA and rif mutants individually temperature-insensitive but temperature-sensitive in combination.

A temperature-sensitive lethal mutant of Escherichia coli has been constructed by combining two temperature-insensitive mutations: a rif180 mutation that modifies RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a strA24 mutation that modifies the ribosomal protein S12. The temperature sensitivity is a property of the combination of these two particular alleles; replacement of either of the alleles relieves the temperature sensitivity. An isogenic strain containing a different strA mutation (i.e., rif180 strA11) is not temperature sensitive. Evidently ribosomes modified by the particular strA24 polymerase altered by the rif180 mutation, which suggests that in vivo there may exist some interaction between structures of ribosomes and the RNA polymerase.

A link between streptomycin and rifampicin mutation.

Introduction of str A mutations frequently make "male" strains of Escherichia coli permissive to bacteriophage T7; certain rif mutations reverse the permissive effect of strA mutation. Permissiveness of the strA mutation is accompanied by enhanced transcription of bacteriophage T7 genome. Introduction of the nonpermissive rif allele to the permissive strA strain reduces or abolishes the transcription of T7 genome. Thus, a link is implied in the functioning of the ribosome and the RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6).

Isolation and characterization of lambda transducing bacteriophages for argF, argI and adjacent genes.

Two genes for ornithinetranscarbamylase exist in strain Escherichia coli K-12, argI, at 85 min, and argF, at 7 min. In an attempt to compare the deoxyribonucleic acid material of these two genes, the lambda transducing phages carrying a portion of the argI region, lambda dvalS argI, lambda pvalS, and lambda dvalS pyrB, and of the argF region, lambda dargF, have been isolated. Their structure, including that of phi 80dargF previously isolated, was studied by the method of heteroduplex mapping. In this paper, the results of this mapping are reported.

A ribonucleoprotein precursor of both the 30S and 50S ribosomal sunbunits of Escherichia coli.

A ribonucleoprotein particle (46S) has been isolated from [3H]uridine pulse-labeled cultures of E. Coli AB301/105. Evidence from pulse chase experiments and from protein analysis suggested that this particle may give rise to both the 30S and 50S ribosomal subunits. Direct deproteinization of the particle yielded 30S RNA, while deproteinization after treatment with a crude RNase III preparation yielded products similar to 23S and 16S RNA. This result is consistent with the idea that the 46S ribonucleoprotein is the in vivo counterpart of 30S RNA, which is the in vitro product obtained after phenol extraction.

Growth of bacteriophages MS2 and T7 on streptomycin-resistant mutants of Escherichia coli.

Streptomycin-resistant mutants of an Hfr strain of Escherichia coli K were examined for their ability to support the growth of male-specific ribonucleic acid phage MS2 and female-specific deoxyribonucleic acid phage T7. Normally, the Hfr strain allows propagation of MS2 and is lysed by it (efficiency of plating equal to 1), whereas the same strain restricts propagation of T7 and is not lysed by it (efficiency of plating smaller than 10-7). Twenty-four isolates out of 26 independently obtained streptomycin-resistant mutants are partially or completely derestricted for propagation of T7; efficiency of plating of T7 in such strains ranges from 10-3-1. Depending on their response to plating of MS2 and T7, the streptomycin-resistant mutants can be divided into four classes. The mutants in all four classes continue to be "male" in conjugation with F- strains. Genetic analysis is presented to show that restriction of MS2, derestriction of T7, and resistance to streptomycin are the pleiotropic effects of a single mutation at the strA locus.

Direct selection of mutants restricting efficiency of suppression and misreading levels in E. coli B.

We describe a method for the direct selection of E. coli mutants restricting efficiency of suppression and misreading levels using a T4-coded nonsense suppressor. One mutant isolated has the phenotype expected for a restrictive mutant and may be ribosomal. Other possibilities are discussed.

A new gene for ribosomal restriction in Escherichia coli.

Two mutations restricting the leakiness of an amber mutant are described. They were selected without the use of streptomycin: one maps in the strA region (at 64 minutes of the current E. coli chromosomal map) but is streptomycin sensitive and the other in the threonine region (at the origin of the map), 23% cotransducible with threonine by P1.

The effects of streptomycin or dihydrostreptomycin binding to 16S RNA or to 30S ribosomal subunits.

Evidence is presented suggesting that streptomycin binds to 16S RNA or to 30S ribosomal subunits at the same topographical site located on the RNA chain. The equally bactericidal dihydrostreptomycin binds to the same site as streptomycin but with lower affinity. The effect of drug binding to 16S RNA (measured by reconstitution inhibition) is readily reversible, while that of drug binding to 30S subunits (measured by misreading) persists after removal of the drug. Binding of the drug is not a necessary and sufficient reason for killing.

Ribosomal assembly influenced by growth in the presence of streptomycin.

Translational leakiness (i.e., nonspecific suppression) of nonsense mutants of bacteriophage T4 is increased in cells of certain streptomycin-resistant strains previously grown in the presence of streptomycin. Concomitantly, ribosomes extracted from these streptomycin-grown cells possess a high level of misreading. Increased suppression ability as well as ribosomes that highly misread accumulate with kinetics expected for a constant differential rate of synthesis of a new product induced by drug action. The misreading ribosomes do not contain appreciable amounts of streptomycin and the misreading property is lost by exposure to high salt concentrations. It is suggested that streptomycin (or dihydrostreptomycin, or paromomycin) induces a reversible modification in 30S subunit assembly without physically participating in the modified structure. The extent of this modification appears dependent upon the strA allele.

The attachment site of streptomycin to the 30S ribosomal subunit.

The 16S RNA dissociated from 30S ribosomal subunits of Escherichia coli strains either sensitive or resistant to streptomycin contains the attachment sites for two streptomycin molecules, as does the undissociated particle from a streptomycin-sensitive strain. Since no streptomycin binds to undissociated 30S subunits from a streptomycin-resistant strain, it is suggested that protein P10, specified by the strA locus-known to be responsible for drug sensitivity-controls the availability to streptomycin of the attachment sites. These sites remain exposed in the strA(+) wild-type, and become masked in strA streptomycin-resistant mutants. The 16S RNA molecule binds streptomycin specifically; it binds two drug molecules in its native state, binds many more after its secondary structure is unfolded by melting out, and again binds two molecules after reannealing. The binding is stable to exhaustive dialysis, but it is reversed by exposure of the streptomycin-RNA complex to high-salt concentration. The complex can not be used to reconstitute functional ribosomes, but the 16S RNA reacquires this property after streptomycin elimination. The biological significance of this stable streptomycin binding is questioned, since in strA mutants exhibiting phenotypic masking, exposure to streptomycin induces a modified 30S behavior that persists even after streptomycin has been dialyzed away, and is reversed only by exposure of the modified RNA to high-salt concentration.

Low activity of -galactosidase in frameshift mutants of Escherichia coli.

16 lac frameshift mutants induced by an acridine derivative, ICR-191D, in E. coli are leaky for beta-galactosidase activity. Activities of all mutants differ from each other and from the wild type in their stability to thermal denaturation. The leakiness is under ribosomal control, since it is strongly reduced by strA restrictive mutations and is restored by ram mutations that reverse restriction. Addition of streptomycin during growth has an effect similar to the presence of the ram mutation. These ribosomal alterations do not modify the thermal stability of the enzyme.It is suggested that the leakiness is due to an infrequent 2- or 4-base reading close to the frameshift mutation site. The possibility that not only the ribosome, but also the reading context in the messenger, plays a role in securing code fidelity is discussed.

Ribosomal alterations controlling alkaline phosphatase isozymes in Escherichia coli.

Different patterns of isozymes were obtained by starch-gel electrophoresis of alkaline phosphatase from Escherichia coli strains differing only by strA or ram mutations, or both, in the 30S ribosomal subunit. The isozyme spread was reduced in strA and increased in ram strains; this strictly parallels the restriction and enhancement of translational ambiguity produced by these mutations. Streptomycin present during growth had an effect similar to ram on both isozymes and ambiguity. The three isozymes analyzed have different N-terminal residues: aspartic acid, valine, and threonine. Different patterns of isozymes were also obtained in a wild-type strain through the specific action of exogenous arginine. A link between the mechanism of the effect of arginine and that of the ribosome is not obvious. The possibility is discussed that in both cases, although by different mechanisms, N-terminals are formed with different sensitivity to limited degradative attack.

Alteration of a 30S ribosomal protein accompanying the ram mutation in Escherichia coli.

The functional peculiarities of ram mutants correlate with an observed alteration in chromatographic mobility of P4(a), a specific protein of the 30S ribosomal subunit. This finding is supported by ribosomal reconstitution experiments. These facts, together with the known location of the ram mutational site in the vicinity of other 30S genetic determinants, suggest that ram is the structural gene for P4(a). The known contrasting roles of ram and strA in determining translational efficiency require that the function of P4(a) should be explained in relation to P10 (the 30S-subunit protein defined by strA). One consequence of altering P4(a), a key protein in ribosome assembly, might be to change the interaction of P10 with the 30S subunit. The functional interrelationship of P4(a) and P10 is discussed in terms of the possible roles of these two proteins in regulating access of tRNA molecules to the decoding site.