Inborn errors of cobalamin metabolism: Effect of cobalamin supplementation in culture on methylmalonyl CoA mutase activity in normal and mutant human fibroblasts

We have examined the effect of addition of hydroxocobalamin to growth medium on the activity of the adenosylcobalamin-requiring enzyme methylmalonyl CoA mutase in normal human fibroblasts and in mutant human fibroblasts derived from patients with inherited methylmalonicacidemia. The mutant cell lines were assigned to four distinct genetic complementation groups (cbl A, cbl B, cbl C, and cbl D), each deficient in some step in the synthesis of adenosylcobalamin from hydroxocobalamin. After control cells were grown in cobalamin-supplemented medium, mutase holoenzyme activity increased markedly in a time- and concentration-dependent fashion. Growth in cobalamin-supplemented medium had no effect on mutase activity in some mutant lines belonging to the cbl B group, while activity increased severalfold in other cbl B mutants and in all cbl A, cbl C, and cbl D mutants examined, although mutase activity was still <10% of control. Comparison of mutase holoenzyme activity and total propionate pathway activity suggests that enhancement of mutase activity in mutant cells after cobalamin supplementation to values 5–10% of control may be sufficient to overcome the inherited metabolic block and to restore total pathway activity to normal.This work was supported in part by a research grant from the National Institutes of Health (AM 12579). H. F. W. is a recipient of a traineeship from the National Institutes of Health (T01-GM02299).     

The Epidemiology of Helicobacter pylori and Public Health Implications

This article presents a review of the literature on the epidemiology and public health implications of Helicobacter pylori infection published from April 2008 through to March 2009. The authors used MeSH terms "Helicobacter infections epidemiology,""Helicobacter infections prevention and control" to search multiple databases (PubMed, Embase, Cochrane, Cochrane Library, EBMR, BIOSIS), and independently searched PubMed using the term "Helicobacter" with "Epidemiology,""Transmission,""Prevalence" or "Environment." Articles without topical relevance were excluded. Two additional papers known to the authors were added. The identified literature is summarized by subtopic: reviews; prevalence; incidence; transmission; risk factors; and public health policy.     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

Four great coccidioidomycologists: William Ophuls (1871-1933), Myrnie Gifford (1892-1966), and Charles Edward Smith (1904-1967) and William A. Winn (1903-1967)

The careers of William Ophuls (1871-1933), Myrnie Gifford (1892-1966), Charles Edward Smith (1904-1967) and William A. Winn (1903-1967) are briefly reviewed, with emphasis on their contributions to knowledge of Coccidioides and coccidioidomycosis. All were students with broad interests, and all were quite willing to suggest new concepts and classifications to replace those of their predecessors.     

How small peptides block and reverse serpin polymerisation

Many of the late-onset dementias, including Alzheimer's disease and the prion encephalopathies, arise from the aberrant aggregation of individual proteins. The serpin family of serine protease inhibitors provides a well-defined structural example of such pathological aggregation, as its mutant variants readily form long-chain polymers, resulting in diseases ranging from thrombosis to dementia. The intermolecular linkages result from the insertion of the reactive site loop of one serpin molecule into the middle strand (s4A) position of the A beta-sheet of another molecule. We define here the structural requirements for small peptides to competitively bind to and block the s4A position to prevent this intermolecular linkage and polymerisation. The entry and anchoring of blocking-peptides is facilitated by the presence of a threonine which inserts into the site equivalent to P8 of s4A. But the critical requirement for small blocking-peptides is demonstrated in crystallographic structures of the complexes formed with selected tri- and tetrapeptides. These structures indicate that the binding is primarily due to the insertion of peptide hydrophobic side-chains into the P4 and P6 sites of s4A. The findings allow the rational design of synthetic blocking-peptides small enough to be suitable for mimetic design. This is demonstrated here with a tetrapeptide that preferentially blocks the polymerisation of a pathologically unstable serpin commonly present in people of European descent.     

Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery

Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro- and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na(+) binding site and that the structure thus may represent the Na(+)-free, catalytically inert "slow" form.     

Uncoupling of genomic and epigenetic signals in the maintenance and inheritance of heterochromatin domains in fission yeast

Many essential aspects of genome function, including gene expression and chromosome segregation, are mediated throughout development and differentiation by changes in the chromatin state. Along with genomic signals encoded in the DNA, epigenetic processes regulate heritable gene expression patterns. Genomic signals such as enhancers, silencers, and repetitive DNA, while required for the establishment of alternative chromatin states, have an unclear role in epigenetic processes that underlie the persistence of chromatin states throughout development. Here, we demonstrate in fission yeast that the maintenance and inheritance of ectopic heterochromatin domains are independent of the genomic sequences necessary for their de novo establishment. We find that both structural heterochromatin and gene silencing can be stably maintained over an ~10-kb domain for up to hundreds of cell divisions in the absence of genomic sequences required for heterochromatin establishment, demonstrating the long-term persistence and stability of this chromatin state. The de novo heterochromatin, despite the absence of nucleation sequences, is also stably inherited through meiosis. Together, these studies provide evidence for chromatin-dependent, epigenetic control of gene silencing that is heritable, stable, and self-sustaining, even in the absence of the originating genomic signals.     

Developmental pathways that generate natural-killer-cell diversity in mice and humans

Natural killer (NK) cells are large granular lymphocytes capable of producing inflammatory cytokines and spontaneously killing malignant, infected or 'stressed' cells. These NK-cell functions are controlled by cell-surface receptors that titrate stimulatory and inhibitory signals. However, we remain puzzled about where and when NK cells develop and differentiate, and this has fuelled the debate over the diversification of the peripheral NK-cell pool: are NK cells functionally homogeneous or are there subsets with specialized effector functions? In this Review, we consider the developmental relationships and biological significance of the diverse NK-cell subsets in mice and humans, and discuss how new humanized mouse models may help to characterize them further.