Mycobacterium tuberculosis infection of a reverse total shoulder arthroplasty: a case report.

We describe the case of a 78-years-old male with dyspnea, inappetence and weight loss over a period of two weeks. The CT scan suggested disseminated tuberculosis and T5-T6 spondylodiscitis. During hospitalization, he developed a left shoulder pain where a reverse total shoulder arthroplasty was implanted 11 years ago. Open debridement and lavage with retention of the implant was performed first and intraveinous antibiotics were administered. 3 months after surgery the patient developed a painful sinus track at the incision site. Resection of the fistula tract, soft tissue debridement and removal of the implants were performed before restarting chemotherapy. As the incidence of reverse total shoulder arthroplasty continues to increase throughout the world, periprosthetic joint infection (PJI) will probably raise as well. Diagnosing and treatment of shoulder PJI with atypical germs remains a challenge and explantation seems to be the safer surgical option to avoid recurrent surgeries on patient with increasing comorbidities.

Immunolocalisation of cytochrome P-450 3A enzymes in human breast carcinoma: relationship with tumour differentiation and steroid receptors.

Cytochrome P-450 3A enzymes belong to the most abundant subfamily of the cytochrome P-450 system. They are predominantly found in the liver where they metabolize numerous drugs and endogenous substances such as oestrogens. However, they are also expressed by normal and tumoural extrahepatic tissues. Accordingly, immunolocalization was assessed in malignant breast tumours (n=32) and normal counterparts, by using a monoclonal antibody that recognizes all human CYP3A proteins. We investigated a potential relation between expression of CYP3A protein expression, the degree of tumour differentiation assessed by the histological grade and the proliferation index assessed by Ki-67 immunostaining. Immunodetection of CYP3A was observed in 27 of the 32 tumours analyzed (84%). A focal staining was also observed in the adjacent normal breast tissue in 33% of the samples, but expression was always fainter than in tumours. A significant negative association was found between CYP3A and the proliferation index, but there was no relation with receptor status or tumour differentiation. While CYP3A protein expression can be found in normal breast tissues, these data highlight higher and more frequent CYP3A in malignant breast cells. Such expression in malignant breast cells appears inversely related to the proliferation index whereas no relation is found with tumour differentiation.

Enzymatic formation of queuosine and of glycosyl queuosine in yeast tRNAs microinjected into Xenopus laevis oocytes. The effect of the anticodon loop sequence.

The eukaryotic tRNA-guanine transglycosylases (queuine insertases) catalyse an exchange of guanine for queuine in position 34, the wobble nucleoside, of tRNAs having a GUN anticodon where N (position 36) stands for A, U, C or G. In tRNAAsp (anticodon QUC) and tRNATyr (anticodon Q psi A) from certain eukaryotic cells, the nucleoside Q-34 is further hypermodified into a glycosylated derivative by tRNA-queuine glycosyltransferase. In order to gain insight into the influence of the nucleosides in position 36, 37 and 38 of an anticodon loop on the potential of a tRNA to become a substrate for the two modifying enzymes, we have constructed several variants of yeast tRNAs in which the normal anticodon has been replaced by one of the synthetic anticodons GUA, GUC, GUG or GUU. In yeast tRNAAsp, the nucleosides 37 (m1G) and 38(C) have also been replaced by an adenosine. These reconstructed chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and tested for their ability to react with the oocyte maturation enzymes. Our results indicate that the nucleosides in positions 36, 37 and 38 influence the efficiencies of conversion of G-34 to Q-34 and of Q-34 to glycosyl Q-34; the importance of their effects are much more pronounced on the glycosylation of Q-34 than on the insertion of queuine. The effect of the nucleoside in position 37 is of particular importance in the case of yeast tRNAAsp: the replacement of the naturally occurring m1G-37 by an unmodified adenosine (as it is in X. laevis tRNAAsp), considerably increases the yield of the glycosylation reaction catalysed by the X. laevis tRNA-queuine glycosyltransferase.

Enzymatic 2'-O-methylation of the wobble nucleoside of eukaryotic tRNAPhe: specificity depends on structural elements outside the anticodon loop.

We have investigated the specificity of the enzyme tRNA (wobble guanosine 2'-O-)methyltransferase which catalyses the maturation of guanosine-34 of eukaryotic tRNAPhe to the 2'-O-methyl derivative Gm-34. This study was done by micro-injection into Xenopus laevis oocytes of restructured yeast tRNAPhe in which the anticodon GmAA and the 3' adjacent nucleotide 'Y' were substituted by various tetranucleotides. The results indicate that the enzyme is cytoplasmic; the chemical nature of the bases of the anticodon and its 3' adjacent nucleotide is not critical for the methylation of G-34; the size of the anticodon loop is however important; structural features beyond the anticodon loop are involved in the specific recognition of the tRNA by the enzyme since Escherichia coli tRNAPhe and four chimeric yeast tRNAs carrying the GAA anticodon are not substrates; unexpectedly, the 2'-O-methylation is not restricted to G-34 since C-34, U-34 and A-34 in restructured yeast tRNAPhe also became methylated. It seems probable that the tRNA (wobble guanosine 2'-O-)methyltransferase is not specific for the type of nucleotide-34 in eukaryotic tRNAPhe; however the existence in the oocyte of several methylation enzymes specific for each nucleotide-34 has not yet been ruled out.

Nucleotide sequences of two serine tRNAs with a GGA anticodon: the structure-function relationships in the serine family of E. coli tRNAs.

We have determined the nucleotide sequence of the major species of E. coli tRNASer and of a minor species having the same GGA anticodon. These two tRNAs should recognize the UCC and UCU codons, the most widely used codons for serine in the highly expressed genes of E. coli. The two sequences differ in only one position of the D-loop. Neither tRNA has a modified adenosine in the position 3'-adjacent to the anticodon. This can be rationalized on the basis of a structural constraint in the anticodon stem and may be related to optimization of the codon-anticodon interaction. Comparison of all E.coli serine tRNAs (and that encoded by bacteriophage T4) reveals characteristic (possibly functional) features. Evolutionary analysis suggests an eubacterial origin of the T4 tRNASer gene and the existence of a recent common ancestor for the tRNASerGGA and tRNASerGUC genes.

The nucleotide sequence of mannosyl-Q-containing tRNAAsp from Xenopus laevis oocytes.

The nucleotide sequence of tRNAAsp from X. laevis oocytes was determined as being: (sequence in text) The tRNA is 75 nucleotides long. This sequence is very similar (75% to 97% identity) to all other eukaryotic tRNAAsp sequenced so far, except for the bovine liver tRNAAsp (32% identity). The relation between the presence of a mannosyl group on queuosine (Q) at position 34 and the nucleotide sequence of the anticodon loop is discussed.

Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence.

We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs. For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U). This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34. Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme. Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all. This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.

Post-transcriptional modification of the wobble nucleotide in anticodon-substituted yeast tRNAArgII after microinjection into Xenopus laevis oocytes.

An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described. Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII. A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase. Such "anticodon-substituted" yeast tRNAArgII are microinjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions. Our results indicate that when adenosine occurs in the wobble position (A34) in yeast tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U. In contrast, when a cytosine (C34) or guanosine (G34) occurs, they are not modified. These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide. In this case, guanosine and uridine were modified while adenosine and cytosine were not.

Site-directed in vitro replacement of nucleosides in the anticodon loop of tRNA: application to the study of structural requirements for queuine insertase activity.

We have investigated the specificity of the enzymes Q-insertase and mannosyl-Q transferase that replace the guanosine at position 34 (wobble base) in the anticodon of several tRNAs by Q or mannosyl-Q derivatives. We have restructured in vitro the normal anticodon of yeast tRNA-Asp-GUC, yeast tRNAArgICG and yeast tRNALeuUAG. With yeast tRNA-Asp-GUC, we have replaced one or several nucleotides in the vicinity of G34 by one of the four canonical nucleotides or by pseudouridylic acid; we have also constructed a tRNAAsp with eight bases instead of seven in the anticodon loop. With yeast tRNAArgICG and yeast tRNALeuUAG, we have replaced their anticodon by the trinucleotide GUC, coding for aspartic acid. The chimerical tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes and after 72 h the amount of Q34 and mannosyl-Q34 incorporated was measured. Our results show that the U33G34U35 sequence, within an anticodon loop of seven bases in chimerical yeast tRNA-Asp-GUC, tRNAArgGUC or tRNALeuGUC, is the main determinant for Q-insertase activity at position 34; the rest of the tRNA sequence has only a slight influence. For mannosyl-Q transferase, however, a much broader structural feature of the tRNA than just the U33G34U35 sequence is important for the efficiency of Q34 transformation into mannosyl-Q34.

Enzymatic replacement in vitro of the first anticodon base of yeast tRNAAsp: application to the study of tRNA maturation in vivo, after microinjection into frog oocytes.

A combination of several enzymes, RNase-T1, nuclease S1, T4-polynucleotide kinase and T4-RNA ligase were used to prepare and modify different fragments of yeast tRNAAsp (normal anticodon G U C). This allowed us to reconstitute, in vitro, a chimeric tRNA that has any of the four bases G, A, U or C, as the first anticodon nucleotide, labelled with (32p) in its 3' position. Such reconstituted (32p) labelled yeast tRNAAsp were microinjected into the cytoplasm or the nucleus of the frog oocyte and checked for their stability as well as for their potential to work as a substrate for the maturation (modifying) enzymes under in vivo conditions. Our results indicate that the chimeric yeast tRNAsAsp were quite stable inside the frog oocyte. Also, the G34 was effectively transformed inside the cytoplasm of frog oocyte into Q34 and mannosyl-Q34; U34 into mcm5s2U and mcm5U. In contrast, C34 and A34 were not transformed at all neither in the cytoplasm nor in the nucleus of the frog oocyte. The above procedure constitutes a new approach in order to detect the presence of a given modifying enzyme inside the frog oocyte; also it provides informations about its cellular location and possibility about its specificity of interaction with foreign tRNA.