Membrane surface properties of sheep erythrocytes,an immunological reagent,after different treatments as reflected by partition in two-polymer aqueous phases

Sheep erythrocytes (E) which, with or without certain treatments, are currently used as “immunological reagents” to detect cells with specific receptors (by rosette-formation) have been partitioned in two-polymer aqueousphase systems selected so as to reflect charge-associatedor lipid-related membrane surface properties. We have found that the partitioning behavior of E is not affected in these phases by reacting the cells with anti-E antibody (either IgG or IgM), forming EA. The additional binding of complement to the cell-antibody complex, forming EAC, results, however, in a marked decrease in the partition coefficient,K. Apparently both the charge-associated and hydrophobic properties reflected by partitioning remain accessible to the phase polymers when the cells are coated with antibody, but are not with the addition of complement. It is interesting that EA can still rosette with T-lymphocytes (14), a property of E, while the additional coating with complement results in EAC which does not appreciably do so (26). Neuraminidase or trypsin treatments of E, which yield Es having quite different rosetting properties with T-lymphocytes (14), cause increasedKs and unchangedKs, respectively, in phases reflecting lipid-related surface properties. Either treatment causes reducedKs of E in charged-phase systems. Neuraminidase treatment also results in a reduced electrophoretic mobility of E, while trypsin treatment is not detectable by cell electrophoresis (25). We are currently studying the possible usefulness of employing cell electrophoresis and cell partitioning in charged-phase systems jointly to obtain information on events occurring at the shear plane versus those occurring deeper in the membrane.     

Biosynthesis of sesquiterpenes in Hymenaea inferred from their quantitative co-occurrence

Leaf pocket resins of 11 species of the tropical arborescent genus Hymenaea are virtually identical qualitatively, but of widely varying quantitative proportions. Within this large range of variability, several strong positive quantitative correlations between resin constituents were found, especially between caryophyllene and β-humulene and between γ-muurolene and δ-cadinene. These data lead to clarification of sesquiterpene biosynthetic routes in Hymenaea. In addition, quantitative relationships found among caryophyllene, α- and β-selinene, γ-muurolene and δ-cadinene are explained only with difficulty by long accepted biosynthetic pathways, and the intermediacy of germacrenes is suggested.     

An immuno-chemo-proteomics method for drug target deconvolution

Chemical proteomics is an emerging technique for drug target deconvolution and profiling the toxicity of known drugs. With the use of this technique, the specificity of a small molecule inhibitor toward its potential targets can be characterized and information thus obtained can be used in optimizing lead compounds. Most commonly, small molecules are immobilized on solid supports and used as affinity chromatography resins to bind targets. However, it is difficult to evaluate the effect of immobilization on the affinity of the compounds to their targets. Here, we describe the development and application of a soluble probe where a small molecule was coupled with a peptide epitope which was used to affinity isolate binding proteins from cell lysate. The soluble probe allowed direct verification that the compound after coupling with peptide epitope retained its binding characteristics. The PKC-alpha inhibitor Bisindolylmaleimide-III was coupled with a peptide containing the FLAG epitope. Following incubation with cellular lysates, the compound and associated proteins were affinity isolated using anti-FLAG antibody beads. Using this approach, we identified the known Bisindolylmaleimide-III targets, PKC-alpha, GSK3-beta, CaMKII, adenosine kinase, CDK2, and quinine reductase type 2, as well as previously unidentified targets PKAC-alpha, prohibitin, VDAC and heme binding proteins. This method was directly compared to the solid-phase method (small molecule was immobilized to a solid support) providing an orthogonal strategy to aid in target deconvolution and help to eliminate false positives originating from nonspecific binding of the proteins to the matrix.     

A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.     

The configuration of the sesquiterpenoid 4-hydroxy-myoporone (athanagrandione)

The conversion of eremoacetal to (?)-1-(furan-3-yl)-4-hydroxy-4,8-dimethylnonane-1,6-dione establishes the configuration of (?)-4-hydroxymyoporone (athanagrandione) as R.     

Structural consequences of metallothionein dimerization: solution structure of the isolated Cd4-alpha-domain and comparison with the holoprotein dimer

The NMR determination of the structure of Cd(7)-metallothionein was done previously using a relatively large protein concentration that favors dimer formation. The reactivity of the protein is also affected under this condition. To examine the influence of protein concentration on metallothionein conformation, the isolated Cd(4)-alpha-domain was prepared from rabbit metallothionein-2 (MT 2), and its three-dimensional structure was determined by heteronuclear, (1)H-(111)Cd, and homonuclear, (1)H-(1)H NMR, correlation experiments. The three-dimensional structure was refined using distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. The backbone superposition of the alpha-domain from rabbit holoprotein Cd(7)-MT 2 and the isolated rabbit Cd(4)-alpha was measured at a RMSD of 2.0 A. Nevertheless, the conformations of the two Cd-thiolate clusters were distinctly different at two of the cadmium centers. In addition, solvent access to the sulfhydryl ligands of the isolated Cd(4)-alpha cluster was 130% larger due to this small change in cluster geometry. To probe whether these differences were an artifact of the structure calculation, the Cd(4)-alpha-domain structure in rabbit Cd(7)-MT 2 was redetermined, using the previously defined set of NOEs and the present calculation protocol. All calculations employed the same ionic radius for Cd(2+) and same cadmium-thiolate bond distance. The newly calculated structure matched the original with an RMSD of 1.24 A. It is hypothesized that differences in the two alpha-domain structures result from a perturbation of the holoprotein structure because of head-to-tail dimerization under the conditions of the NMR experiments.     

Hydroxylated analogues of the orally active broad spectrum antifungal, Sch 51048 (1), and the discovery of posaconazole [Sch 56592; 2 or (S,S)-5

As part of a detailed study, the syntheses, biological activities, and pharmacokinetic properties of hydroxylated analogues of the previously described broad spectrum antifungal agents, Sch 51048 (1), Sch 50001 (3), and Sch 50002 (4), are described. Based on an overall superior profile, one of the alcohols, Sch 56592 (2), was selected for clinical studies.