The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli.

The structural gene (glnA) encoding the glutamine synthetase (GS) of the extremely thermophilic eubacterium Thermotoga maritima has been cloned on a 6.0 kb HindIII DNA fragment. Sequencing of the region containing the glnA gene (1444 bp) showed an ORF encoding a polypeptide (439 residues) with an estimated mass of 50,088 Da, which shared significant homology with the GSI sequences of other Bacteria (Escherichia coli, Bacillus subtilis) and Archaea (Pyrococcus woesei, Sulfolobus solfataricus). The T. maritima glnA gene was expressed in E. coli, as shown by the ability to complement a glnA lesion in the glutamine-auxotrophic strain ET8051. The recombinant GS has been partially characterized with respect to the temperature dependence of enzyme activity, molecular mass and mode of regulation. The molecular mass of the Thermotoga GS (590,000 Da), estimated by gel filtration, was compatible with a dodecameric composition for the holoenzyme, as expected for a glutamine synthetase of the GSI type. Comparison of the amino acid sequence of T. maritima GS with those from thermophilic and mesophilic micro-organisms failed to detect any obvious features directly related to thermal stability.

Anti-HBV neonatal immunization with recombinant vaccine. Part II. Molecular basis of the impaired alloreactivity.

HLA study was performed in 9 absolute non-responder (serum titre of anti-HBsAg < 2 mIU ml-1) and 8 hyporesponder (serum antibody level between 2 and 9.9 mIU ml-1) babies who underwent, in neonatal period, HBV vaccination with Engerix B recombinant vaccine. The investigation pointed out that many of these subjects carry HLA haplotypes classically involved in autoimmune diseases: namely HLADR7; DQ2, DR4; DQ8 and DR3; DQ2. The genomic typing for DRB1, DRB3, DRB4, DRB5, DQA1, DQB1 and DPB1 genes revealed an increased frequency of the DRB1*0701; DQA1*0201; DQB1*0201 haplotype (23.5 vs 9.9% of the controls) and of DPB*0201 allele (42.3 vs 13.2% of controls). The polymorphism of Bf, C4A and C4B complement serum components, recognized as important "immune-function-related genes", pointed out an increased frequency of the null allele C4AQ0 (34.3 vs 6.8% of the controls) stressing the role of C4A serum complement component in response to foreign peptide. The immunogenetic investigation has been extended to 23 responder babies (titre of anti-HBsAg > 50 mIU ml-1), vaccinated with the same trial as the poor responders. The HLA frequencies observed in this group were comparable to those of control population and, with respect to the HLA markers cited above, absolutely different from the non/hyporesponder infants. From the HLA class II sequence analysis in the group of poor-responder babies some characteristics, peculiar to autoimmune diseases, have been observed: the majority of the infants showed at least an arginine at the 52 residue of the alpha chain of DQ molecule and a non-aspartic acid at the 57 position of the DQ beta chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of allogeneic bone marrow transplantation from an HLA-identical sibling or a matched unrelated donor in the treatment of children with juvenile chronic myeloid leukaemia.

Seven children (age range 1.8-11 years) with juvenile chronic myelomonocytic leukaemia (JCML) received an allogeneic bone marrow transplantation (BMT), four from an HLA-identical sibling and three from a matched unrelated donor. In the four children transplanted using an HLA-identical sibling, conditioning regimen included busulfan (BU), cyclophosphamide (CY) and melphalan (L-PAM), whereas graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine-A (Cs-A). The preparative regimen was well tolerated and all patients engrafted promptly. None of the patients have relapsed and all four children remain in haematological remission after an observation time of 7, 24, 25 and 48 months, respectively. Of the three children given BMT from an unrelated volunteer, one was < 2 years of age and she received the BU/CY/L-PAM regimen. In view of the increased risk of graft rejection described in patients transplanted from unrelated donors, we chose to prepare the other two patients with fractionated total body irradiation (TBI), thiotepa and CY. Cs-A, short-term methotrexate and Campath-1G in vivo were employed to prevent GVHD in this group of patients. Graft failure with autologous reconstitution of haemopoiesis occurred in the child given the chemotherapy-based regimen. One of the two girls given TBI relapsed after BMT; therefore only one of the three patients who received a marrow transplant from a matched unrelated donor survives in complete haematological remission 10 months after BMT. Our study suggests that the conditioning regimen we employed for allogeneic BMT from a compatible sibling is an effective means of eradicating the leukaemic clone. In our experience, results obtained using unrelated donors are less satisfactory and, at present, the use of such donors seems to be riskier and associated with a lower success rate as compared with BMT from an HLA-identical sibling.