Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen.

Previous work demonstrated that human furin is a predominantly Golgi membrane-localized endoprotease that can efficiently process precursor proteins at paired basic residues (-Lys-Arg- or -Arg-Arg-) in transfected cells. Anion-exchange chromatography of culture supernatant from cells expressing a soluble truncated form of human furin resulted in a greatly enriched preparation of the endoprotease (approximately 70% pure as determined by protein staining). Enzymatic studies show that furin is a calcium-dependent (K0.5 = 200 microM) serine endoprotease which has greater than 50% of maximal activity between pH 6.0 and 8.5. The inhibitor sensitivity of furin suggests that it is similar to, yet distinct from, other calcium-dependent proteases. Evidence that furin may require a P4 Arg in fluorogenic peptide substrates suggested that this enzyme might cleave the protective antigen (PA) component of anthrax toxin at the sequence -Arg-Lys-Lys-Arg-. Indeed, PA was cleaved by purified furin at the proposed consensus site (-Arg-X-Lys/Arg-Arg decreases-) at a rate (8 mumol/min/mg total protein) 400-fold higher than that observed with synthetic peptides. In addition, the processing of mutant PA molecules with altered cleavage sites suggests that furin-catalyzed endoproteolysis minimally requires an -Arg-X-X-Arg- recognition sequence for efficient cleavage. Together, these results support the hypothesis that furin processes protein precursors containing this cleavage site motif in the exocytic pathway and in addition, raises the possibility that the enzyme also cleaves extracellular substrates, including PA.     

Comparative life history and respiratory activity of “wild” and colonized Caribbean fruit flies [Dipt.: Tephritidae]

Insects that are colonized for use in biological control programs must be behaviorally functional with respect to target field populations. To quantify adaptation during colonization and identify some of the behavioral changes that occur during the process, comparable colonies of field-collected and laboratory-adapted Caribbean fruit flies,Anastrepha suspensa (Loew) were monitored through 5 generations for fertility of eggs, weights and yields of pupae, and viability, sex ratio, insemination frequency, fecundity and CO² production of adults. After five generations, the wild strain still produced 57% fewer eggs, required 6–7 extra days per cycle (16% longer), and produced 16% less CO² per generation     

SCA8 RAN polySer protein preferentially accumulates in white matter regions and is regulated by eIF3F

Spinocerebellar ataxia type 8 (SCA8) is caused by a bidirectionally transcribed CTG·CAG expansion that results in the in vivo accumulation of CUG RNA foci, an ATG‐initiated polyGln and a polyAla protein expressed by repeat‐associated non‐ATG (RAN) translation. Although RAN proteins have been reported in a growing number of diseases, the mechanisms and role of RAN translation in disease are poorly understood. We report a novel toxic SCA8 polySer protein which accumulates in white matter (WM) regions as aggregates that increase with age and disease severity. WM regions with polySer aggregates show demyelination and axonal degeneration in SCA8 human and mouse brains. Additionally, knockdown of the eukaryotic translation initiation factor eIF3F in cells reduces steady‐state levels of SCA8 polySer and other RAN proteins. Taken together, these data show polySer and WM abnormalities contribute to SCA8 and identify eIF3F as a novel modulator of RAN protein accumulation.     

Intermolecular complementation achieves high-specificity tumor targeting by anthrax toxin

Anthrax toxin protective antigen (PrAg) forms a heptamer in which the binding site for lethal factor (LF) spans two adjacent monomers. This suggested that high cell-type specificity in tumor targeting could be obtained using monomers that generate functional LF-binding sites only through intermolecular complementation. We created PrAg mutants with mutations affecting different LF-binding subsites and containing either urokinase plasminogen activator (uPA) or matrix metalloproteinase (MMP) cleavage sites. Individually, these PrAg mutants had low toxicity as a result of impaired LF binding, but when administered together to uPA- and MMP-expressing tumor cells, they assembled into functional LF-binding heteroheptamers. The mixture of two complementing PrAg variants had greatly reduced toxicity in mice and was highly effective in the treatment of aggressive transplanted tumors of diverse origin. These results show that anthrax toxin, and by implication other multimeric toxins, offer excellent opportunities to introduce multiple-specificity determinants and thereby achieve high therapeutic indices.     

Distribution of an endogenous lectin in the developing chick optic tectum

We determined the cellular localization of an endogenous lectin at various times during the development of a well-characterized region of chick brain, the optic tectum. This lectin is a carbohydrate-binding protein that interacts with lactose and other saccharides, undergoes striking changes in specific activity with development, and has previously been purified by affinity chromatography from extracts of embryonic chick brain and muscle. Cellular localization in the tectum was done by indirect immunofluoresecent staining, using immunoglobulin G derived from an antiserum raised against pure lectin. No lectin was detectable in the optic tectum examined at 5 days of embryonic development. From approximately 7 days of development, neuronal cell bodies and fibers were labeled by the antibody; and extracts of tectum contained hemagglutination activity that could be inhibited by lactose or by the antiserum. Lectin remained present in many tectal neuronal layers after hatching; but in 2-month-old chicks it was sparse or absent in most of the tectum except for prominent labeling of fibers in the stratum album centrale. The initial appearance of lectin in the optic tectum was not dependent on innervation by optic nerve fibers since bilateral enucleation during embryogenesis did not affect it. Lectin was detectable on the surface of embryonic optic tectal neurons dissociated with a buffer containing EDTA.     

Large-scale structural changes accompany binding of lethal factor to anthrax protective antigen: a cryo-electron microscopic study

Anthrax toxin (AT), secreted by Bacillus anthracis, is a three-protein cocktail of lethal factor (LF, 90 kDa), edema factor (EF, 89 kDa), and the protective antigen (PA, 83 kDa). Steps in anthrax toxicity involve (1) binding of ligand (EF/LF) to a heptamer of PA63 (PA63h) generated after N-terminal proteolytic cleavage of PA and, (2) following endocytosis of the complex, translocation of the ligand into the cytosol by an as yet unknown mechanism. The PA63h.LF complex was directly visualized from analysis of images of specimens suspended in vitrified buffer by cryo-electron microscopy, which revealed that the LF molecule, localized to the nonmembrane-interacting face of the oligomer, interacts with four successive PA63 monomers and partially unravels the heptamer, thereby widening the central lumen. The observed structural reorganization in PA63h likely facilitates the passage of the large 90 kDa LF molecule through the lumen en route to its eventual delivery across the membrane bilayer.     

An antibody-based microarray assay for small RNA detection

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 µl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.