Crystallisation and preliminary crystallographic analysis of P1 nuclease from Penicillium citrinum

P1 nuclease, a zinc-dependent single-strand specific endonuclease from Penicillium citrinum, has been crystallized in three different space groups using either ammonium sulphate or polyethylene glycol 4000 as the precipitating agent. The crystals diffract to between 3 A and 2.2 A. A 4.5 A electron density map has been calculated for a tetragonal crystal form, based on a platinum derivative, and was improved by solvent flattening. The boundaries of the two molecules in the asymmetric unit are clearly visible in most regions and the presence of rod-like density features are indicative of a rather high alpha-helix content. The highest density peaks in the map were identified as a trinuclear zinc cluster present in each monomer by a difference Fourier of an EDTA-soaked crystal.     

Assignment of Alu-repetitive sequences to large restriction fragments from human chromosomes 6 and 22

We have employed a pulsed field gel electrophoresis and Alu hybridization approach for identification of large restriction fragments on chromosome 6 and 22. This technique allows large portions of selected human chromosomes to be visualized as discrete hybridization signals. Somatic cell hybrid DNA which contains chromosome 6 or chromosome 22 was restricted with either Notl or Mlul. The restriction fragments were separated by pulsed field gel electrophoresis (PFGE) and hybridized against an Alu repetitive sequence (Blur 8). The hybridization signals result in a fingerprint-like pattern which is unique for each chromosome and each restriction enzyme. In addition, a continuous pattern of restriction fragments was demonstrated by gradually increasing puls times. This approach will also be suitable to analyze aberrant human chromosomes retained in somatic cell hybrids and can be used to analyze flow sorted human chromosomes. To this end, our method provides a valuable alternative to standard cytogenetic analysis.     

The primary structure of monkey pituitary growth hormone

Monkey growth hormone (MGH) has been purified by reverse-phase high-performance liquid chromatography (HPLC). Tryptic digests of MGH were separated by HPLC and paper electrophoresis. From amino acid composition, from NH2-terminal residue and sequence analyses of these tryptic peptides, and from their alignment with those of human growth hormone, the primary structure of MGH was proposed. There are only four residues which are different in these two growth hormone molecules.     

Specific polyclonal antibodies to the carboxyl terminus of [Met]enkephalin-Arg6-Gly7-Leu8

The octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu was recently isolated from bovine adrenal chromaffin granules and serves as a marker for proenkephalin from which it is derived. Polyclonal antisera which are highly specific for the carboxyl terminus have been raised against the synthetic peptide. The only significant cross-reactivity was with the 18.2-k Da and 5.3-k Da enkephalin-containing peptides (EC peptides) which contain the octapeptide at their carboxyl termini and the [des-Tyr] and [des-Tyr-Gly] congeners of the octapeptide. Extracts of bovine adrenal medulla and rat spinal cord were shown to contain significant amounts of the octapeptide, the two larger EC peptides, and the two smaller congeners.     

Two human TNF receptors have similar extracellular, but distinct intracellular, domain sequences

Tumor necrosis factor (TNF) is a cytokine with a wide range of biological activities in inflammatory and immunologic responses. These activities are mediated by specific cell surface receptors of 55 kDa and 75 kDa apparent molecular masses. A 75-kDa TNF receptor cDNA was isolated using partial amino acid sequence information and the polymerase chain reaction (PCR). When expressed in COS-1 cells, the cDNA transfers specific TNF-binding properties comparable to those of the native receptor. The predicted extracellular region contains four domains with characteristic cysteine residues highly similar to those of the 55-kDa TNF receptor, the nerve growth factor (NGF) receptor, and the CDw40 and OX40 antigens. The consensus sequence of the TNF receptor extracellular domains also has similarity to the cysteine-rich sequence motif LIM. In marked contrast to the extracellular regions, the intracellular domains of the two TNF receptors are entirely unrelated, suggesting different modes of signaling and function.     

New and selective ryanodine receptor activators for insect control

Diamide insecticides have emerged as one of the most promising new classes of insecticide chemistry owing to their excellent insecticidal efficacy and high margins of mammalian safety. Chlorantraniliprole and flubendiamide, the first two insecticides from this class, demonstrate exceptional activity across a broad range of pests in the order Lepidoptera. This chemistry has been confirmed to control insects via activation of ryanodine receptors which leads to uncontrolled calcium release in muscle. The high levels of mammalian safety are attributed to a strong selectivity for insect over mammalian receptors.     

Cholesterol conjugation potentiates the antiviral activity of an HIV immunoadhesin

Immunoadhesins are engineered proteins combining the constant domain (Fc) of an antibody with a ligand‐binding (adhesion) domain. They have significant potential as therapeutic agents, because they maintain the favourable pharmacokinetics of antibodies with an expanded repertoire of ligand‐binding domains: proteins, peptides, or small molecules. We have recently reported that the addition of a cholesterol group to two HIV antibodies can dramatically improve their antiviral potency. Cholesterol, which can be conjugated at various positions in the antibody, including the constant (Fc) domain, endows the conjugate with affinity for the membrane lipid rafts, thus increasing its concentration at the site where viral entry occurs. Here, we extend this strategy to an HIV immunoadhesin, combining a cholesterol‐conjugated Fc domain with the peptide fusion inhibitor C41. The immunoadhesin C41‐Fc‐chol displayed high affinity for Human Embryonic Kidney (HEK) 293 cells, and when tested on a panel of HIV‐1 strains, it was considerably more potent than the unconjugated C41‐Fc construct. Potentiation of antiviral activity was comparable to what was previously observed for the cholesterol‐conjugated HIV antibodies. Given the key role of cholesterol in lipid raft formation and viral fusion, we expect that the same strategy should be broadly applicable to enveloped viruses, for many of which it is already known the sequence of a peptide fusion inhibitor similar to C41. Moreover, the sequence of heptad repeat‐derived fusion inhibitors can often be predicted from genomic information alone, opening a path to immunoadhesins against emerging viruses. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.