Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana.

The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphate-mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.     

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array.     

Voltage dependence of Na-Ca exchanger conformational currents.

Properties of a transient current (Icont) believed to reflect a conformational change of the Na-Ca exchanger molecules after Ca2+ binding were investigated. Intracellular Ca2+ concentration jumps in isolated cardiac myocytes were generated with flash photolysis of caged Ca2+ dimethoxynitrophenamine, and membrane currents were simultaneously measured using the whole-cell variant of the patch-clamp technique. A previously unresolved shallow voltage dependence of Icont was revealed after developing an experimental protocol designed to compensate for the photoconsumption of the caged compound. This voltage dependence can be interpreted to reflect the distribution of Na-Ca exchanger conformational states with the Ca2+ binding site exposed to the inside of the cell immediately before the flash. Analysis performed by fitting a Boltzmann distribution to the observed data suggests that under control conditions most exchanger molecules reside in states with the Ca2+ binding site facing the outside of the cell. Dialysis of the cytosol with 3',4'-dichlorobenzamil, an organic inhibitor of the Na-Ca exchange, increased the magnitude of Icont and changed the voltage dependence, consistent with a parallel shift of the charge/voltage curve. This shift may result from intracellular DCB interfering with an Na(+)-binding or Na(+)-translocating step. These observations are consistent with Icont arising from a charge movement mediated by the Na-Ca exchanger molecules after binding of Ca2+.     

Epstein-Barr virus transformation of human B lymphocytes despite inhibition of viral polymerase.

Epstein-Barr virus transformed human lymphocytes despite the presence of up to 500 microM acyclovir [9-(2-hydroxyethoxymethyl)guanine], a viral DNA polymerase inhibitor. The transformed cells contained multiple Epstein-Barr virus genome copy numbers. Functional viral DNA polymerase is probably not required for cell transformation and the initial amplification of the viral genome.     

Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.     

Heterozygote advantage in Tay-Sachs carriers?

Chi-square analyses of new data as well as data previously reported by Myrianthopoulos have shown that grandparents of Tay-Sachs carriers die from proportionally the same causes as grandparents of noncarriers. It is unlikely that there is any advantage to being a Tay-Sachs carrier insofar as resistance to tuberculosis is concerned. Our results are further evidence to support Fraikor's claim that the high carrier frequency of the allele in Ashkenazi Jews is probably caused by a combination of founder effect, genetic drift, and differential immigration patterns.