A mitochondrial DNA D loop insertion detected almost exclusively in non-replicating tissues with maternal inheritance across three generations.

Muscle biopsy identified a possibly pathogenic, mitochondrial DNA D-loop insertion, in each of 5 family members from two generations, that was otherwise undetectable in most other tissues. The tissue specific regulation of heteroplasmy is reflected in an age related increase in muscle heteroplasmy level, across the pedigree. This latter finding is in keeping with previous reports (e.g. T408A, C16327) but differs in having a very high muscle heteroplasmy level, and appears maternally transmitted.

Drug-sensitive DNA polymerase δ reveals a role for mismatch repair in checkpoint activation in yeast.

We have used a novel method to activate the DNA damage S-phase checkpoint response in Saccharomyces cerevisiae to slow lagging-strand DNA replication by exposing cells expressing a drug-sensitive DNA polymerase δ (L612M-DNA pol δ) to the inhibitory drug phosphonoacetic acid (PAA). PAA-treated pol3-L612M cells arrest as large-budded cells with a single nucleus in the bud neck. This arrest requires all of the components of the S-phase DNA damage checkpoint: Mec1, Rad9, the DNA damage clamp Ddc1-Rad17-Mec3, and the Rad24-dependent clamp loader, but does not depend on Mrc1, which acts as the signaling adapter for the replication checkpoint. In addition to the above components, a fully functional mismatch repair system, including Exo1, is required to activate the S-phase damage checkpoint and for cells to survive drug exposure. We propose that mismatch repair activity produces persisting single-stranded DNA gaps in PAA-treated pol3-L612M cells that are required to increase DNA damage above the threshold needed for checkpoint activation. Our studies have important implications for understanding how cells avoid inappropriate checkpoint activation because of normal discontinuities in lagging-strand replication and identify a role for mismatch repair in checkpoint activation that is needed to maintain genome integrity.

Lipoamide dehydrogenase mediates retention of coronin-1 on BCG vacuoles, leading to arrest in phagosome maturation.

Mycobacterium tuberculosis evades the innate antimicrobial defenses of macrophages by inhibiting the maturation of its phagosome to a bactericidal phagolysosome. Despite intense studies of the mycobacterial phagosome, the mechanism of mycobacterial persistence dependent on prolonged phagosomal retention of the coat protein coronin-1 is still unclear. The present study demonstrated that several mycobacterial proteins traffic intracellularly in M. bovis BCG-infected cells and that one of them, with an apparent subunit size of M(r) 50,000, actively retains coronin-1 on the phagosomal membrane. This protein was initially termed coronin-interacting protein (CIP)50 and was shown to be also expressed by M. tuberculosis but not by the non-pathogenic species M. smegmatis. Cell-free system experiments using a GST-coronin-1 construct showed that binding of CIP50 to coronin-1 required cholesterol. Thereafter, mass spectrometry sequencing identified mycobacterial lipoamide dehydrogenase C (LpdC) as a coronin-1 binding protein. M. smegmatis over-expressing Mtb LpdC protein acquired the capacity to maintain coronin-1 on the phagosomal membrane and this prolonged its survival within the macrophage. Importantly, IFNgamma-induced phagolysosome fusion in cells infected with BCG resulted in the dissociation of the LpdC-coronin-1 complex by a mechanism dependent, at least in part, on IFNgamma-induced LRG-47 expression. These findings provide further support for the relevance of the LpdC-coronin-1 interaction in phagosome maturation arrest.

Nonswelling macroporous synbeads for improved efficiency of solid-phase biotransformations.

An application of novel, highly porous nonswelling resins (Synbeads) for enzymatic catalysis on solid supports is reported. These new resins combine easy handling of the beads, chemical stability, improved accessibility of proteins and higher productivity relative to swelling polymers. The present study demonstrates that the resin porosity greatly affects the efficiency in solid-phase biotransformations and that Synbead resins are valuable alternatives to swelling polymers for solid-phase chemistry and biocatalysis. The present study investigates the influence of key parameters, such as porosity and reactive functional-group density, on the reaction efficiency.

Reversible immobilization of invertase on Sepabeads coated with polyethyleneimine: optimization of the biocatalyst's stability.

Invertase from S. cerevisiae has been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE-agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers-octamers at low pH value). The optimal enzyme preparation remained fully active after a 15-day incubation at 50 degrees C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 degrees C higher than that of soluble enzyme.

Epoxy sepabeads: a novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment.

Sepabeads-EP (a new epoxy support) has been utilized to immobilize-stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment. These supports are very robust and suitable for industrial purposes. Also, the internal geometry of the support is composed by cylindrical pores surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme), and it has a very high superficial density of epoxy groups (around 100 micromol/mL). These features should permit a very intense enzyme-support interaction. However, the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol. By using conventional immobilization protocols (neutral pH values, nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA. However, when using a more sophisticated three-step immobilization/stabilization/blockage procedure, the Sepabeads derivative was hundreds-fold more stable than Eupergit C derivatives. The protocol used was as follows: (i) the enzyme was first covalently immobilized under very mild experimental conditions (e.g., pH 7.0 and 20 degrees C); (ii) the already immobilized enzyme was further incubated under more drastic conditions (higher pH values, long incubation periods, etc.) in order to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; (iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support. This third point was found to be critical for obtaining very stable enzymes: derivatives blocked with mercaptoethanol were much less stable than derivatives blocked with glycine or other amino acids. This was attributed to the better masking of the hydrophobicity of the support by the amino acids (having two charges).