Characterization of bacteriophages infecting Streptomyces erythreus and properties of phage-resistant mutants.

Three bacteriophages infecting Streptomyces erythreus, called G3, G4 and G5, were isolated and characterized. They contain double-stranded linear DNA molecules with cohesive ends. The restriction map of G3 DNA (48 kilobases long) for four restriction endonucleases and that of G4 DNA (43 kilobases long) for seven restriction endonucleases are reported. Restriction analysis and hybridization experiments showed that G3 and G4 share little DNA homology, while G4 and G5 are apparently identical except for an additional EcoRI site present in G5. The region containing this EcoRI site has been mapped on G4 DNA. Microbiological and serological data showed that G5 is very similar to G4. G3- and G4-resistant mutants of S. erythreus PS1 were isolated. The screening of phage-resistant mutants showed a high frequency of strains with increased erythromycin production. The mechanism of phage resistance of strain PS3 (G3 resistant) and of strain PS16 (G4 resistant) was examined. The DNA of the resistant strains contains no phage DNA, ruling out lysogeny as a cause of phage resistance. Transfection of strains PS1, PS3, and PS16 with DNA of the three phages showed the same efficiency, indicating that resistance is at the level of the bacterial wall.     

Tight linkage of glnA and a putative regulatory gene in Rhizobium leguminosarum.

Rhizobium leguminosarum, biovar viceae, strain RCC1001 contains two glutamine synthetase activities, GSI and GSII. We report here the identification of glnA, the structural gene for GSI. A 2 kb fragment of DNA was shown to complement the Gln- phenotype of Klebsiella pneumoniae glnA mutant strains. DNA sequence analysis revealed an open reading frame (ORF) of 469 codons specifying a polypeptide of 52,040 daltons. Its deduced amino acid sequence was found to be highly homologous to other glutamine synthetase sequences. This ORF was expressed in Escherichia coli minicells and the corresponding polypeptide reacted with an antiserum raised against GSI. Upstream of glnA we found an ORF of 111 codons (ORF111) preceded by the consensus sequence for an ntrA-dependent promoter. Minicells experiments showed a protein band, with a molecular weight in good agreement with that (10,469) deduced from the nucleotide sequence. On the basis of homology studies we discuss the possibility that the product of ORF111 is equivalent to the PII protein of E. coli and plays a similar role in regulation of nitrogen metabolism.     

Impaired mitochondrial calcium uptake caused by tacrolimus underlies beta-cell failure

Background

One of the most common side effects of the immunosuppressive drug tacrolimus (FK506) is the increased risk of new-onset diabetes mellitus. However, the molecular mechanisms underlying this association have not been fully clarified.

Methods

We studied the effects of the therapeutic dose of tacrolimus on mitochondrial fitness in beta-cells.

Results

We demonstrate that tacrolimus impairs glucose-stimulated insulin secretion (GSIS) in beta-cells through a previously unidentified mechanism. Indeed, tacrolimus causes a decrease in mitochondrial Ca²⁺ uptake, accompanied by altered mitochondrial respiration and reduced ATP production, eventually leading to impaired GSIS.

Conclusion

Our observations individuate a new fundamental mechanism responsible for the augmented incidence of diabetes following tacrolimus treatment. Indeed, this drug alters Ca²⁺ fluxes in mitochondria, thereby compromising metabolism-secretion coupling in beta-cells.

     

Effect of pregnancy on serum cytokines in SLE patients

Introduction

The aim of this study was to evaluate an extensive panel of cytokines involved in immune regulation during pregnancy in patients with systemic lupus erythematosus (SLE) and in healthy women.

Methods

A total of 47 consecutive successful pregnancies in 46 SLE patients and 56 pregnancies in 56 matched healthy subjects, as controls, were prospectively studied. Serum interleukin (IL)-1-α, IL-1-β, IL-2, IL-6, IL-8, IL-10, IL-12p70, interferon (INF)-γ and tumor necrosis factor (TNF)-α were detected in sera obtained at the first and third trimester of pregnancy by a highly sensitive, multiplexed sandwich ELISA.

Results

Medians (pg/ml) of serum levels of most helper T (Th)1-type cytokines were significantly lower in the third trimester compared with those observed in the first trimester of pregnancy in healthy women: INF-γ 2.0 vs 3.4, TNF-α 10.2 vs 11.5, IL-1-α 0.9 vs 1.1, IL-1-β 0.6 vs 1.0, IL-2 3.0 vs 3.5, and IL-12p70 4.9 vs 5.6 (P-values < 0.02 for all). By contrast, only the IL-1-α serum levels were lower in the third trimester compared with the first trimester in SLE patients (P = 0.006). IFN-γ/IL-6 and IFN-γ/IL-10 ratios were higher in controls than in SLE (P = 0.002, and P = 0.001, respectively); moreover, they were significantly reduced in the third compared to the first trimester of pregnancy in healthy women, but not in SLE.

Conclusions

In SLE patients, Th1/Th2 cytokine serum level ratio does not decrease during pregnancy progression as much as in healthy pregnant women. This could account for the observation of a low frequency of disease flares in the third trimester of gestation.     

The glnB gene of Rhizobium leguminosarum biovar viceae

Abstract An open-reading frame (ORF111) upstream of the glutamine synthetase I structural gene ( glnA ) in Rhizobium leguminosarum biovar viceae encodes a protein which is highly homologous to the P_II protein (encoded by glnB ) of enteric bacteria. ORF111 was cloned in a number of different plasmid vectors and shown to complement a K. pneumoniae glnB mutant. We propose that ORF111 encodes the P_II protein of R. leguminosarum and that it should be designated glnB .