Glycogen metabolism loss: a common marker of parasitic behaviour in bacteria?

We searched 55 completely sequenced bacterial genomes for glycogen synthesis and degradation enzymes. A significant proportion of these bacteria appears to lack glycogen metabolism capability. Interestingly, these bacteria are parasitic, symbiotic or fastidious (i.e. difficult to culture outside their normal environment). It is suggested that the lack of bacterial glycogen metabolism is a trait associated with parasitic behaviour in bacteria.     

Extensive expansion of primary human gamma delta T cells generates cytotoxic effector memory cells that can be labeled with Feraheme for cellular MRI

Gamma delta T cells (GDTc) comprise a small subset of cytolytic T cells shown to kill malignant cells in vitro and in vivo. We have developed a novel protocol to expand GDTc from human blood whereby GDTc were initially expanded in the presence of alpha beta T cells (ABTc) that were then depleted prior to use. We achieved clinically relevant expansions of up to 18,485-fold total GDTc, with 18,849-fold expansion of the Vδ1 GDTc subset over 21 days. ABTc depletion yielded 88.1 ± 4.2 % GDTc purity, and GDTc continued to expand after separation. Immunophenotyping revealed that expanded GDTc were mostly CD27-CD45RA- and CD27-CD45RA+ effector memory cells. GDTc cytotoxicity against PC-3M prostate cancer, U87 glioblastoma and EM-2 leukemia cells was confirmed. Both expanded Vδ1 and Vδ2 GDTc were cytotoxic to PC-3M in a T cell antigen receptor- and CD18-dependent manner. We are the first to label GDTc with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles for cellular MRI. Using protamine sulfate and magnetofection, we achieved up to 40 % labeling with clinically approved Feraheme (Ferumoxytol), as determined by enumeration of Perls’ Prussian blue-stained cytospins. Electron microscopy at 2,800× magnification verified the presence of internalized clusters of iron oxide; however, high iron uptake correlated negatively with cell viability. We found improved USPIO uptake later in culture. MRI of GDTc in agarose phantoms was performed at 3 Tesla. The signal-to-noise ratios for unlabeled and labeled cells were 56 and 21, respectively. Thus, Feraheme-labeled GDTc could be readily detected in vitro via MRI.     

Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.     

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

In vitro differentiation of inflammatory and non-inflammatory states of the synovial fluid by magnetic protonic relaxation

The differentiation between inflammatory and non inflammatory states has been performed using Nuclear Magnetic Resonance (NMR) in vitro by measuring relaxation times T1 and T2 in 84 synovials fluids obtained from various rheumatologic diseases. The results show that the T1/T2 ratio is more sensitive to distinguish these two situations rather than the isolated T1 or T2 values. In particular, high values of T1/T2 ratio are found in septic arthritis.