The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding

The solution structure of the (6)F1(1)F2(2)F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'.     

The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells

We describe a novel murine progenitor cell population localised to a previously uncharacterised region between sebaceous glands and the hair follicle bulge, defined by its reactivity to the thymic epithelial progenitor cell marker MTS24. MTS24 labels a membrane-bound antigen present during the early stages of hair follicle development and in adult mice. MTS24 co-localises with expression of alpha6-integrin and keratin 14, indicating that these cells include basal keratinocytes. This novel population does not express the bulge-specific stem cell markers CD34 or keratin 15, and is infrequently BrdU label retaining. MTS24-positive and -negative keratinocyte populations were isolated by flow cytometry and assessed for colony-forming efficiency. MTS24-positive keratinocytes exhibited a two-fold increase in colony formation and colony size compared to MTS24-negative basal keratinocytes. In addition, both the MTS24-positive and CD34-positive subpopulations were capable of producing secondary colonies after serial passage of individual cell clones. Finally, gene expression profiles of MTS24 and CD34 subpopulations were compared. These results showed that the overall gene expression profile of MTS24-positive cells resembles the pattern previously reported in bulge stem cells. Taken together, these data suggest that the cell-surface marker MTS24 identifies a new reservoir of hair follicle keratinocytes with a proliferative capacity and gene expression profile suggestive of progenitor or stem cells.     

NADPH dependent activation of microsomal glutathione transferase 1

Microsomal glutathione transferase 1 (MGST1) can become activated up to 30-fold by several mechanisms in vitro (e.g. covalent modification by reactive electrophiles such as N-ethylmaleimide (NEM)). Activation has also been observed in vivo during oxidative stress. It has been noted that an NADPH generating system (g.s.) can activate MGST1 (up to 2-fold) in microsomal incubations, but the mechanism was unclear. We show here that NADPH g.s treatment impaired N-ethylmaleimide activation, indicating a shared target (identified as cysteine-49 in the latter case). Furthermore, NADPH activation was prevented by sulfhydryl compounds (glutathione and dithiothreitol). A well established candidate for activation would be oxidative stress, however we could exclude that oxidation mediated by cytochrome P450 2E1 (or flavine monooxygenase) was responsible for activation under a defined set of experimental conditions since superoxide or hydrogen peroxide alone did not activate the enzyme (in microsomes prepared by our routine procedure). Actually, the ability of MGST1 to become activated by hydrogen peroxide is critically dependent on the microsome preparation method (which influences hydrogen peroxide decomposition rate as shown here), explaining variable results in the literature. NADPH g.s. dependent activation of MGST1 could instead be explained, at least partly, by a direct effect observed also with purified enzyme (up to 1.4-fold activation). This activation was inhibited by sulfhydryl compounds and thus displays the same characteristics as that of the microsomal system. Whereas NADPH, and also ATP, activated purified MGST1, several nucleotide analogues did not, demonstrating specificity. It is thus an intriguing possibility that MGST1 function could be modulated by ligands (as well as reactive oxygen species) during oxidative stress when sulfhydryls are depleted.     

Mutants partially defective in excision repair at five autosomal loci in Drosophila melanogaster

Primary cell cultures derived from mutants in seventeen different genes were analyzed for their ability to excise pyrimidine dimers from DNA. Five of these mutagen-sensitive mutants [mus(2)205^A1, mus(3)302^D1, mus(3) 304^D3, mus(3)306^D1, mus(3)308^D2] display a significantly reduced excision capacity relative to control cultures. In addition, two of the five [mus(3)306^D1, mus(3)308^D2] are defective in the accumulation of single-strand breaks normally seen after ultraviolet irradiation. This study, therefore, brings the total number of Drosophila mutants known to be defective in excision repair to seven. The results are discussed relative to other genetic and biochemical properties of these mutants. This work is dedicated to Professor W. Beermann whose own contributions were instrumental in focusing a modern analysis of the eukaryotic genome on the diptera. Those of us who benefitted so much from his personal guidance recognize that we did so as a result of some sacrifice on his part. One of Boyd's contemporaries in Tübingen once remarked: “It's terrifying to think what Professor Beermann could do if he were in the lab full time.”     

Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

Background

A number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment.

Results

Using a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP.

Conclusions

We have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain.     

REPRODUCTIVE ATTRITION IN THE RARE CHAPARRAL SHRUB FREMONTODENDRON DECUMBENS LLOYD (STERCULIACEAE)

We examined reproductive attrition in Fremontodendron decumbens to characterize sexual reproduction in this rare California shrub. Reproductive individuals produced an average of 2,900 flower buds in a season, with no significant difference in bud production between two seasons. Because of intense insect predation, <;2% of initiated flower buds became mature fruits. A threefold decrease in predation of flower buds between seasons resulted in an increase in seed output the second season, indicating that seed production was partially predator-limited. Most seeds (97.8%) were dormant due to an impermeable seed coat. Breaking of the coat, mechanically or by heat, allowed high levels of germination. Chamise charate and ash added to the potting medium resulted in the highest level of germination and emergence. Rodents were more important than birds as seed predators, destroying 90% of seeds under parent shrub canopies within 8–10 months. Seeds already integrated into the seed bank were comparatively safe from predation, relative to newly added seeds. If predation was prevented, seeds were long-lived under field conditions (>;80% survived after 5.75 years). Most seedlings produced in unburned chaparral by planting heat-treated seeds in openings between shrubs were destroyed by predators (rodents and insects). All seedlings that escaped predation died during the summer drought. We concluded that sexual reproduction was limited by (in order of importance): 1) lack of fire, 2) predehiscence predation by insects, and 3) postdehiscence predation by rodents. Size distributions from two populations revealed that, despite the apparent absence of sexual reproduction in unbumed chaparral, two unbumed sites contained a large proportion of individuals in small size classes. Excavation of several small individuals demonstrated they were sprouts from the roots of nearby larger shrubs. Because asexual reproduction by rootsprouting circumvents the high attrition of sexual reproductive effort on unbumed sites, rootsprouting may be a significant reproductive strategy of some ‘sprouter’ species in chaparral.