Genetic analysis of sterile mutants in the dpy-5 unc-13 (I) genomic region of Caenorhabditis elegans

Essential genes were identified in the 1.5-map unit dpy-5 unc-13 region of chromosome I in the Caenorhabditis elegans genome by rescuing lethal mutations using the duplication sDp2. In this paper, we report the mapping and complementation testing of lethal mutations, 45 of which identify 18 new, essential genes. This analysis brings the number of essential genes defined by the sDp2 rescue of lethal mutants to 97; 64 of these map between dpy-5 and unc-13. 61% of these essential genes are identified by more than one allele. Positioning of the mutations was done using the breakpoints of six duplications. The mutant phenotypes of 14 loci essential for fertility were characterized by Nomarski microscopy and DAPI staining. None of the mutants were rescued by wild-type male sperm. The cytological data showed that four genes produced mutants with defects in gonadogenesis, let-395, let-603, let-605 and let-610. Mutations in seven genes, let-355, let-367, let-384, let-513, let-544, let-545 and let-606, affected germ cell proliferation or gametogenesis. Mutants for the remaining three genes, let-370, let-599 and let-604, produced eggs that failed to develop or hatch, thereby acting as maternal effect lethals. We observed a nonrandom distribution of arrest phenotypes with regard to map position. Received: 8 May 1996 / Accepted : 27 January 1997     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

Targeting age‐specific changes in CD4+ T cell metabolism ameliorates alloimmune responses and prolongs graft survival

Age impacts alloimmunity. Effects of aging on T‐cell metabolism and the potential to interfere with immunosuppressants have not been explored yet. Here, we dissected metabolic pathways of CD4⁺ and CD8⁺ T cells in aging and offer novel immunosuppressive targets. Upon activation, CD4⁺ T cells from old mice failed to exhibit adequate metabolic reprogramming resulting into compromised metabolic pathways, including oxidative phosphorylation (OXPHOS) and glycolysis. Comparable results were also observed in elderly human patients. Although glutaminolysis remained the dominant and age‐independent source of mitochondria for activated CD4⁺ T cells, old but not young CD4⁺ T cells relied heavily on glutaminolysis. Treating young and old murine and human CD4⁺ T cells with 6‐diazo‐5‐oxo‐l‐norleucine (DON), a glutaminolysis inhibitor resulted in significantly reduced IFN‐γ production and compromised proliferative capacities specifically of old CD4⁺ T cells. Of translational relevance, old and young mice that had been transplanted with fully mismatched skin grafts and treated with DON demonstrated dampened Th1‐ and Th17‐driven alloimmune responses. Moreover, DON diminished cytokine production and proliferation of old CD4⁺ T cells in vivo leading to a significantly prolonged allograft survival specifically in old recipients. Graft prolongation in young animals, in contrast, was only achieved when DON was applied in combination with an inhibition of glycolysis (2‐deoxy‐d‐glucose, 2‐DG) and OXPHOS (metformin), two alternative metabolic pathways. Notably, metabolic treatment had not been linked to toxicities. Remarkably, immunosuppressive capacities of DON were specific to CD4⁺ T cells as adoptively transferred young CD4⁺ T cells prevented immunosuppressive capacities of DON on allograft survival in old recipients. Depletion of CD8⁺ T cells did not alter transplant outcomes in either young or old recipients. Taken together, our data introduce an age‐specific metabolic reprogramming of CD4⁺ T cells. Targeting those pathways offers novel and age‐specific approaches for immunosuppression.     

Activation of superoxide production and differential exocytosis in polymorphonuclear leukocytes by cytochalasins A,B, C,D and E: Effects of various ions

All of the common cytochalasins activate superoxide anion release and exocytosis of β-N-acetylglucosaminidase and lysozyme from guinea-pig polymorphonuclear leukocytes (neutrophils) incubated in a buffered sucrose medium. Half-maximal activation of both processes is produced by approx. 2 μM cytochalasin A, C >μM cytochalasin B ? 4–5 μM cytochalasin D, E. While maximal rates of O₂^? release and extents of exocytosis require extracellular calcium (1–2 mM), replacing sucrose with monovalent cation chlorides is inhibitory to neutrophil activation by cytochalasins. Na⁺, K⁺ or choline inhibited either cytochalasin B- or E-stimulated O₂^? production with IC₅₀ values of 5–10 mM and inhibition occurs whether Cl^?, NO₃^? or SCN^? is the anion added with Na⁺ or K⁺. Release of β-N-acetylglucosaminidase in control or cytochalasin B-stimulated cells is inhibited by NaCl (IC₅₀ ≈ 10 mM), while cytochalasin E-stimulated exocytosis is reduced less and K⁺ or choline chloride are ineffective in inhibiting either cytochalasin B- or E-stimulated exocytosis. Release of β-glucuronidase, myeloperoxidase or acid phosphatase from neutrophils incubated in buffered sucrose is not stimulated by cytochalasin B. Stimulation of either O₂^? or β-N-acetylglucosaminidase release by low concentrations of cytochalasin A is followed by inhibition of each at higher concentrations. It appears that all cytochalasins can activate both NAD(P)H oxidase and selective degranulation of neutrophils incubated in salt-restricted media and that differential inhibition of these two processes by monovalent cations and/or anions is produced at some step(s) subsequent to cytochalasin interaction with the cell.     

Soil and Fertilizer Amendments and Edge Effects on the Floral Succession of Pulverized Fuel Ash

Plots of fresh pulverized fuel ash (PFA, an industrial waste) were inoculated with soils from existing PFA sites and fertilizers in a factorial design, then left unmanaged for 12 years during which time the floral development and soil chemistry were monitored annually. For the first 3 years, the site supported a sparse mix of chenopods (including the scarce Chenopodium glaucum ) and halophytes. As salinity declined, ruderals, legumes, and grasses plus the fire-site moss Funaria hygrometrica colonized, followed by Festuca arundinacea grassland (NVC community MG12) and Hippophae rhamnoides scrub. Dactylorhiza incarnata (orchidacea) appeared after 7 years, but only in plots that had received soil from existing orchid colonies. Four years later, a larger second generation of Dactylorhiza appeared, but only in the central zone of the site where vegetation was thinnest. By year 12, the site was dominated by coarse grasses and scrub, with early successional species persisting only in the sparsely vegetated center, where nitrate levels were lowest. This edge effect is interpreted as centripetal encroachment, a process of potentially wider concern for the conservation of low-fertility habitat patches. Overall, seed bank inoculation seems to have introduced few but desirable species ( D. incarnata , Pyrola rotundifolia , some halophytes, and annuals), whereas initial application of organic fertilizer had long-lasting (≥10 years) effects on cover and soil composition.     

Metabolic plasticity drives development during mammalian embryogenesis

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