Sequence-specific peptide cleavage catalyzed by an antibody

Monoclonal antibodies have been induced that are capable of catalyzing specific hydrolysis of the Gly-Phe bond of peptide substrates at neutral pH with a metal complex cofactor. The antibodies were produced by immunizing with a Co(III) triethylenetetramine (trien)-peptide hapten. These antibodies as a group are capable of binding trien complexes of not only Co(III) but also of numerous other metals. Six peptides were examined as possible substrates with the antibodies and various metal complexes. Two of these peptides were cleaved by several of the antibodies. One antibody was studied in detail, and cleavage was observed for the substrates with the trien complexes of Zn(II), Ga(III), Fe(III), In(III), Cu(II), Ni(II), Lu(III), Mg(II), or Mn(II) as cofactors. A turnover number of 6 x 10(-4) per second was observed for these substrates. These results demonstrate the feasibility of the use of cofactor-assisted catalysis in an antibody binding site to accomplish difficult chemical transformations.     

Uptake of isolated chloroplasts by mammalian cells

Mouse fibroblasts (L cells) in suspension culture incorporated isolated chloroplasts of spinach and African violets and isolated mitochondria of chicken liver. The organelles resided in the cell cytoplasm and were not contained in vacuoles or digestion vesicles. Green cells divided like normal cells. Green chloroplasts were followed for five cell generations or 5 days, at which time hybrid cells were greatly outnumbered by nongreen progeny cells. The ingested chloroplasts retained their structural integrity as determined by electron microscopy of organelles and hybrid cells and by analysis of photochemical activity and DNA in chloroplasts reisolated from cells after 1 or 2 days in culture.     

Regulation of Wnt signaling and embryo patterning by an extracellular sulfatase

The developmental signaling functions of cell surface heparan sulfate proteoglycans (HSPGs) are dependent on their sulfation states. Here, we report the identification of QSulf1, the avian ortholog of an evolutionarily conserved protein family related to heparan-specific N-acetyl glucosamine sulfatases. QSulf1 expression is induced by Sonic hedgehog in myogenic somite progenitors in quail embryos and is required for the activation of MyoD, a Wnt-induced regulator of muscle specification. QSulf1 is localized on the cell surface and regulates heparan-dependent Wnt signaling in C2C12 myogenic progenitor cells through a mechanism that requires its catalytic activity, providing evidence that QSulf1 regulates Wnt signaling through desulfation of cell surface HSPGs.     

Organization of iron-sulfur clusters in respiratory complex I

Complex I of respiratory chains plays a central role in bioenergetics and is implicated in many human neurodegenerative diseases. An understanding of its mechanism requires a knowledge of the organization of redox centers. The arrangement of iron-sulfur clusters in the hydrophilic domain of complex I from Thermus thermophilus has been determined with the use of x-ray crystallography. One binuclear and six tetranuclear clusters are arranged, maximally 14 angstroms apart, in an 84-angstrom-long electron transfer chain. The binuclear cluster N1a and the tetranuclear cluster N7 are not in this pathway. Cluster N1a may play a role in the prevention of oxidative damage. The structure provides a framework for the interpretation of the large amounts of data accumulated on complex I.     

Galactosyl diglycerides: their possible function in Euglena chloroplasts

Illumination of euglenas grown in the dark induces the formation of chloroplasts characterized by the simultaneous appearance of chlorophyll and galactosyl diglycerides in a relatively fixed ratio. The fatty acyl chains of the galactosyl diglycerides are constructed so that they can provide a stable lock-and-key fit with the phytol chains of chlorophyll in such a way as to localize the porphyrin structures of chlorophyll and space them for efficient photoreception. Light-starved photobiotic euglenas show chloroplast shrinkage with a concurrent partial loss of galactosyl diglycerides.     

SIR1, an upstream component in auxin signaling identified by chemical genetics

Auxin is a plant hormone that regulates many aspects of plant growth and development. We used a chemical genetics approach to identify SIR1, a regulator of many auxin-inducible genes. The sir1 mutant was resistant to sirtinol, a small molecule that activates many auxin-inducible genes and promotes auxin-related developmental phenotypes. SIR1 is predicted to encode a protein composed of a ubiquitin-activating enzyme E1-like domain and a Rhodanese-like domain homologous to that of prolyl isomerase. We suggest a molecular context for how the auxin signal is propagated to exert its biological effects.     

Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling

The ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) plays a central role in the cellular response to changes in oxygen availability. pVHL binds to HIF only when a conserved proline in HIF is hydroxylated, a modification that is oxygen-dependent. The 1.85 angstrom structure of a 20-residue HIF-1alpha peptide-pVHL-ElonginB-ElonginC complex shows that HIF-1alpha binds to pVHL in an extended beta strand-like conformation. The hydroxyproline inserts into a gap in the pVHL hydrophobic core, at a site that is a hotspot for tumorigenic mutations, with its 4-hydroxyl group recognized by buried serine and histidine residues. Although the beta sheet-like interactions contribute to the stability of the complex, the hydroxyproline contacts are central to the strict specificity characteristic of signaling.     

Selective cleavage of human DNA: RecA-assisted restriction endonuclease (RARE) cleavage

Current methods for sequence-specific cleavage of large segments of DNA are severely limited because of the paucity of possible cleavage sites. A method is described whereby any Eco RI site can be targeted for specific cleavage. The technique is based on the ability of RecA protein from Escherichia coli to pair an oligonucleotide to its homologous sequence in duplex DNA and to form a three-stranded complex. This complex is protected from Eco RI methylase; after methylation and RecA protein removal, Eco RI restriction enzyme cleavage was limited to the site previously protected from methylation. When pairs of oligonucleotides are used, a specific fragment can be cleaved out of genomes. The method was tested on lambda phage, Escherichia coli, and human DNA. Fragments exceeding 500 kilobases in length and yields exceeding 80 percent could be obtained.     

Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes

Single base substitutions can be detected and localized by a simple and rapid method that involves ribonuclease cleavage of single base mismatches in RNA:DNA heteroduplexes. A 32P-labeled RNA probe complementary to wild-type DNA is synthesized in vitro and annealed to a test DNA containing a single base substitution. The resulting single base mismatch is cleaved by ribonuclease A, and the location of the mismatch is then determined by analyzing the sizes of the cleavage products by gel electrophoresis. Analysis of every type of mismatch in many different sequence contexts indicates that more than 50 percent of all single base substitutions can be detected. The feasibility of this method for localizing base substitutions directly in genomic DNA samples is demonstrated by the detection of single base mutations in DNA obtained from individuals with beta-thalassemia, a genetic disorder in beta-globin gene expression.     

Sulfate Reduction in Soil: Effects of Redox Potential and pH

Reduction of sulfate to sulfide in waterlogged soils was dependent on soil redox potential and pH. Little or no sulfide accumulated with a redox potential above -150 millivolts, or with a pH outside the range 6.5 to 8.5.     

Sex differences in electric signaling in an electric fish

The electric discharge of Sternopygus macrurus is distinctly different from the discharges of ten sympatric species of electric fish in Guyana, South America. Sternopygus is the first known example of a fish with sexually different electric discharges. Males and females differ in the steady-state frequency of their discharges, and males produce variations in their discharge during courtship. Playback experiments demonstrate that species and sex differences in electric discharges have communicative significance.     

Selective cleavage of D-Ala-D-Lac by small molecules: re-sensitizing resistant bacteria to vancomycin

Pathogenic enterococci are becoming resistant to currently available antibiotics, including vancomycin, the drug of last resort for Gram-positive infections. Enterococci pose a significant public health threat, not least because of the risk of transferring vancomycin resistance to the ubiquitous Staphylococcus aureus. Vancomycin resistance is manifested by cell wall peptidoglycan precursors with altered termini that cannot bind the antibiotic. Small molecules with well-oriented nucleophile-electrophile assembly and complementary chirality to the peptidoglycan termini were identified as catalytic and selective cleavers of the peptidoglycan precursor depsipeptide. These molecules were tested in combination with vancomycin and were found to re-sensitize vancomycin-resistant bacteria to the antibiotic.     

Site of biosynthesis of galactolipids in spinach chloroplasts

The envelope of the spinach chloroplast is the site of galactolipid synthesis.     

Slaty cleavage: incipient occurrences in the deep sea

Highly deformed Pleistocene mudstones from the inner wall of the Aleutian Trench and from the continental rise of the Gulf of Mexico show incipient slaty cleavage defined by the orientation of platy and elongate detrital minerals parallel to the axial surfaces of folds.